Deprotection method and resin removal method in solid-phase reaction for peptide compound or amide compound, and method for producing peptide compound

ABSTRACT

The present inventors found that peptide compounds/amide compounds in which the protecting groups of interest are removed and/or which are removed from resins for solid-phase synthesis can be produced without main chain damage by contacting starting peptide compounds/amide compounds with silylating agents.

TECHNICAL FIELD

The present invention relates to methods of deprotection of peptidecompounds or amide compounds and methods of removing such compounds fromresins in solid-phase reactions, and methods of producing peptidecompounds.

BACKGROUND ART

Peptides are molecules in which many amino acids are linked together.Extensive research has been conducted not only on the synthesis ofpeptides produced by organisms, but also on peptides with artificiallydesigned structures and desired functions (NPL 1).

Peptides can be produced by linking multiple amino acids by repeatingthe steps of (i) activating the carboxyl group of an N-terminallyprotected, C-terminally unprotected amino acid with a condensing agentor the like to provide an active ester, (ii) reacting the active esterwith an N-terminally unprotected peptide to provide a peptide elongatedwith the amino acid, and (iii) removing the N-terminal protecting groupfrom the elongated peptide (NPL 2).

Peptide production methods are roughly classified into solid-phasemethods and liquid-phase methods. Solid-phase methods are performed byallowing the C-terminus of an amino acid or a derivative thereof (aprotected amino acid) or a peptide or a derivative thereof to bind to aresin for solid-phase synthesis that contains a chlorotrityl group, abenzyl group, or the like, such as CTC resin, Wang resin, or SASRINresin, and performing coupling reaction between the amino group of theresulting amino acid or peptide, which serves as a nucleophile, and thecarbonyl group of an N-terminally protected peptide or amino acid, whichserves as an electrophile. Liquid-phase methods are performed bycoupling reaction between the carbonyl group of an N-terminallyprotected amino acid or peptide as an electrophile and the amino groupof an amino acid or peptide as a nucleophile. When such couplingreactions may lead to undesired transformation of functional groups inthe amino acid or peptide side chains, protecting groups must bepreviously introduced into these side chains. Protecting groups that canbe removed under acidic conditions such as Boc, t-Bu, or trityl groupsare generally used to protect the C-terminus of amino acids or peptides,the N-terminus of amino acids or peptides, and the side chains of aminoacids or peptides. In resin removal reactions to cleave peptides fromresin for solid-phase synthesis to which they are bound, resins forsolid-phase synthesis that are removable under acidic conditions such asCTC resin, Wang resin, and SASRIN resin are generally used (NPL 4).

Protecting groups that can be removed under acidic conditions alsoinclude 3,5-dimethoxyphenylisopropoxycarbonyl (Ddz), benzyloxycarbonyl(Cbz), benzyl (Bn), and cyclohexyl (cHx) groups (NPL 4 and NPL 5). Underthe conditions of their deprotection reactions or the resin removalreactions, side reactions may occur such as cleavage of amide bonds inpeptides and unintended transformation of functional groups in aminoacid side chains, resulting in by-product peptides having unintendedsequences. Accordingly, there is a need to provide target peptideswithout reactions damaging the peptide main chain, such as amide bondcleavage reactions and rearrangement reactions of the peptide mainchain.

The above-described protecting groups that can be removed under acidicconditions are often removed by treatment with hydrochloric acid,sulfuric acid, methanesulfonic acid, or TFA (NPL 2, NPL 3, and NPL 4).TFA is usually used in resin removal reaction in solid-phase synthesis(NPL 6). However, it has been described that amide bonds may be cleavedeven under acidic conditions using TFA, the mildest acid among them (NPL7).

It has also been described that an attempt to solve the problem of amidebond cleavage was made by conducting the reactions under diluted TFAconditions and the Boc deprotection reactions under mild conditions;however, the problem was not solved under both conditions (NPL 8, PTL1). Methods of removing protecting groups from protected amino acidsusing less acidic TFE have also been reported (NPL 5, PTL 1). In thesedocuments, the reaction conversion rate is low, and it is thereforenecessary to add more acid or conduct the reactions under severerreaction conditions such as elevated reaction temperatures in order toadvance the reactions. However, when reaction conditions includingaddition of acid or elevation of reaction temperatures are applied to apeptide, there is a concern that the main chain of the peptide may bedamaged. Actually, when Boc-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl was reactedwith hydrogen chloride in TFE to perform Boc-deprotection reaction, mainchain cleavage was observed before the Boc deprotection reaction wascompleted.

It has also been reported that amide bond cleavage was successfullysuppressed by conducting the reaction under low-temperature conditionsand stopping the reaction before side reactions occurred (NPL 9).However, this technique requires strict control of the reaction time,and it is easily presumed that such control is challenging in massproduction, where it is difficult to stop reactions in a short time.

As a deprotection method under acidic conditions, an attempt to inhibitside reactions between side chains and t-Bu cation generatedconcomitantly in the removal of Boc or t-Bu group has been made byadding a cation scavenger (NPL 10).

In this case, the deprotection under acidic conditions was performedwith sulfur-containing additives such as dimethyl sulfide andthioanisole to scavenge t-Bu cations derived from t-Bu groups, but itreportedly caused amide bond cleavage as a side reaction, resulting inreduced yield and purity (NPL 7).

A method of allowing thioanisole and TfOH to act in a TFA solution orallowing thioanisole and trimethylsilyl bromide or trimethylsilyltriflate to act in a TFA solution has also been reported (NPL 12).

However, many sulfur-containing additives, represented by dimethylsulfide and thioanisole, have offensive odor and thus their effects onmanufacturing workers are a concern. In addition, it is problematic thatremoval of such additives requires removal steps by columnchromatography, which is unsuitable for mass production.

It has also been reported that even when using water as a cationscavenger, peptide chain cleavage was increased (NPL 11).

Deprotection methods using silicon-containing additives instead ofsulfur-containing additives as cation scavengers have been reported,where N-Boc protected compounds containing amide bonds are treated withcation scavengers such as triisopropylsilane (iPr3SiH), for example, ata ratio of TFA:iPr3SiH:H2O=95:2.5:2.5 (NPL 13) or at a ratio ofTFA:iPr3SiH:H2O:PhOH=1000:50:67:50 (NPL 14). However, as for thesemethods, only conditions difficult to achieve in mass production aredescribed, such as performing the treatment for a short period of timeof several minutes to suppress amide bond cleavage. In fact, whenBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MePhe-MeAla-Leu-MeLeu-Thr(OBn)-MeGly-OAllylwas subjected to Boc deprotection reaction by applying the conditions ofNPL 13 and NPL 14, cleavage of the amide bond between MePhe and MeAlawas observed before removal of the Boc group was completed. The cleavagerate was also increased over time.

As described above, when deprotection reactions and resin removalreactions in solid-phase synthesis are carried out under acidicconditions, they may involve not only desired deprotection or resinremoval reactions but also damage to the peptide main chain such as thecleavage of amide bonds that constitute the peptide main chain and therearrangement of functional groups of amino acid side chains to the mainchain. Production of by-products due to such main chain damage is knownto reduce the yield and purity of target products and is a problem forefficient synthesis of peptides.

In the meantime, it has also been reported that the structural featuresof peptides are associated with the stability or damageability of amidebonds.

For example, distortion of amide bonds is known to cause increased acidinstability of the amide bonds (NPL 15 and NPL 16).

Furthermore, in addition to the bond distortion, in the case of peptideamide bonds, the types of the amino acid side chains, the modes ofsubstitution of the nitrogen atoms, the number of amino acid residues inthe peptide, and the amino acid sequence affect the modes ofintramolecular hydrogen bonds and the stable conformation of thepeptide. Depending on such conformational changes, carbonyl oxygens maybecome more basic and susceptible to protonation (NPL 18 and NPL 19). Itis known that in some cases amide bonds are damaged by formation ofoxazolone (NPL 17).

It has also been reported that peptides containing N-Me amino acids,which are amino acids with the nitrogen atom methylated, may undergoprogressive amide bond cleavage under acidic conditions (NPL 11, NPL 17,and NPL 20). Moreover, it is known that when a peptide having an N-Meamino acid at the C-terminus is subjected to TFA acidic conditions, theamide bond between the first and second residues of the peptide may becleaved, resulting in a by-product peptide lacking the C-terminalresidue (NPL 21). Thus, it is known that amide bonds containing N-Megroups in N-Me amino acids are readily cleaved.

Additionally, it is known that peptides having a sequence with two ormore consecutive N-Me amino acid residues are often labile to acid andundergo amide bond cleavage during resin removal reactions insolid-phase synthesis (NPL 11). Thus, it is known that amide bondscontaining N-Me groups in N-Me amino acids and/or amide bonds containingcarbonyl groups in N-Me amino acids are readily cleaved.

Peptides containing Asp or Gln are also labile to acid in asequence-dependent manner (NPL 22 and NPL 23). For example, it is knownthat when a peptide sequence having Asp is treated with acid such ashydrogen fluoride and methanesulfonic acid, the carbonyl group of itsside chain is nucleophilically attacked by the nitrogen atom of a mainchain amide, yielding aspartimide as a by-product (NPL 23). Thus, it isknown that amide bonds are readily cleaved in sites containing an aminoacid having a specific side chain, even if the amino acid isN-unsubstituted.

When this aspartimide intermediate is hydrolyzed, rearrangementby-products with altered amide structures and main chain-cleavedby-products are generated, thereby reducing the yield and purity of thetarget product. Formation of aspartimides is notable when peptidescontaining a sequence such as Asp(OBn)-Gly, Asp(OBn)-Ser, Asp(OBn)-Thr,Asp(OBn)-Asn, or Asp(OBn)-Gln are deprotected under acidic conditions.For example, it is known that when Boc-Phe-Asp(OBn)-Asn-Ala-OBn isdeprotected by methanesulfonic acid, it is mostly converted toaspartimides and does not have a desired amino acid sequence, i.e., mainchain-damaging reaction occurs (NPL 24).

It is also known that when peptide derivatives containing Asp-Pro intheir sequences are subjected to acidic conditions such astrifluoroacetic acid, hydrofluoric acid, formic acid, and acetic acid,the side chain of Asp is reacted with the nitrogen atom of Pro as in theabove-described side reaction and the amide bond is thus cleaved (NPL 25and NPL 26).

There are some reports of methods for avoiding side reactions indeprotection reaction that are caused by the amino acidsequence-dependent instability of amide bonds against acid.

For example, a method of suppressing the formation of aspartimides indeprotection and resin removal reactions of aspartamide derivatives isknown, where the carboxyl group of the side chain of aspartic acid isprotected with a cyclohexyl group so that the nitrogen atoms ofmain-chain amides do not proceed with nucleophilic attacks under thedeprotection conditions for the protecting group of interest. Thismethod has been reported to significantly suppress the formation ofaspartimides as compared with the case of using a benzyl protectinggroup as a side-chain protecting groups (NPL 27). However, subsequentremoval of the cyclohexyl group requires highly corrosive, highly acidicconditions such as hydrofluoric acid, and it is readily presumed thatboth the reaction and the post-treatment cannot be conductedconveniently.

A method of treatment with 1 M trimethylsilyl bromide/TFA in thepresence of thioanisole is also known (NPL 28). However, this conditionis disadvantageous in that it requires the use of a solvent amount ofTFA and the use of thioanisole, which is unsuitable for mass productionas described above. Furthermore, it has been described that by-productaspartimides are significantly increased when trimethylsilyl triflate isused instead of trimethylsilyl bromide, suggesting that trimethylsilylbromide is superior to trimethylsilyl triflate.

There is a known method in which Boc removal reaction of peptides andt-Bu removal reaction of esters are performed in the presence of atertiary amine and in the presence of trimethylsilyl triflate orTBDMSOTf. A known example is to use 2,6-lutidine as a tertiary amine andperform N-Boc removal under TMSOTf/2,6-lutidine conditions in thepresence of other functional groups, thereby removing Boc groupsselectively (NPL 29, NPL 30, and NPL 31). In another known example,reactions for Boc removal and removal from resin for solid-phasesynthesis, which may occur at the same time under acidic conditionsusing TFA, are performed under trimethylsilyl triflate/2,6-lutidineconditions to achieve Boc removal reaction selectively (NPL 32 and 33).These methods have also been applied to compounds having amide bonds,but not intended to avoid damaging amide bonds.

Selective deprotection of t-Bu esters in the presence of TBS and TIPSgroups and thioacetal has been performed under trimethylsilyltriflate/2,6-lutidine conditions (NPL 34). Removal of t-Bu from peptidet-Bu esters using this technique has also been reported. For example, itis known that deprotection reaction of t-Bu esters was performed underTMSOTf/2,6-lutidine conditions (NPL 35 and NPL 36). This case, however,is also not intended to avoid amide bond cleavage.

It is known that when trimethylsilyl triflate/2,6-lutidine is used forN-Boc removal reactions in the total synthesis of a natural peptidecompound that provides complex mixtures upon treatment with protonicacid, the reactions preferentially yield the target product whilesuppressing production of by-products (NPL 37). However, side reactionsreferred to therein are not described in detail, and these reactionconditions were not examined at all for the general versatility and thepossibility of avoiding amide bond damage. Moreover, reagents are neededin large excess (20 equivalents or more), leaving problems from theviewpoint of the generality of applicable substrates and manufacturingcost. In this case, trimethylsilyl triflate/2,6-lutidine was merelyapplied to compounds having amide bonds, and it is not mentioned whetheror not the side reactions were caused by amide bond cleavage.

As described above, treatment under acidic conditions using Bronstedacids represented by trifluoroacetic acid is generally used in reactionsof cleaving peptides from peptide-loaded resins (resin removalreactions) to provide elongated peptides in the solid-phase methods, andin reactions of removing protecting groups removable under acidicconditions among N-terminal, C-terminal, and amino acid side-chainprotecting groups in both the solid-phase methods and the liquid-phasemethods. There are also known examples in which deprotection reactionsare conducted in the presence of Lewis acids. However, despite the knownfact that treatment of peptides containing unnatural amino acidsrepresented by N-Me amino acids with conventional methods to performresin removal reaction or deprotection reaction results in the intendedreaction accompanied with progression of main-chain cleavage orrearrangement (main-chain damage) of the peptides and requires largequantities of reagents, no solution for these problems is known (NPL 20,NPL 22, and NPL 37).

In particular, since studies of unnatural artificial peptides oftenencounter unexpected side reactions when conventional synthesis methodsare used, it is extremely important to develop industrially efficienttechniques to synthesize unnatural artificial peptides and theirderivatives. However, in producing such unnatural amino acid-containingpeptides, only methods of preparing conventional peptides composed ofnatural amino acids have been used. There is no known efficientsynthesis method that focuses on and solves the problems with theproduction of peptides containing unnatural amino acids, in particularthe specific problem of main chain-damage caused in deprotection orresin removal reaction.

CITATION LIST Patent Literature

-   [PTL 1] WO 2014/033466

Non-Patent Literature

-   [NPL 1] Future Med. Chem., 2009, 1, 1289-1310.-   [NPL 2] Amino Acids, Peptides and Proteins in Organic Chemistry:    Building Blocks, Catalysis and Coupling Chemistry, Volume 3, 2011-   [NPL 3] Amino Acids, 2018, 50, 39-68.-   [NPL 4] Chem. Rev., 2009, 109, 2455-2504.-   [NPL 5] Russ. J. Bioorg. Chem. 2016, 42, 143-152.-   [NPL 6] J. Comb. Chem. 2002, 4, 1-16.-   [NPL 7] J. Am. Chem. Soc. 1999, 121, 6786-6791.-   [NPL 8] Org. Lett. 2012, 14, 612-615.-   [NPL 9] Eur. J. Med. Chem. 2000, 35, 599-618.-   [NPL 10] J. Chem. Soc. D, 1970, 0, 406b-407.-   [NPL 11] J. Peptide Res. 2005, 65, 153-166.-   [NPL 12] J. Chem. Soc. Chem. Commun. 1987, 274-275.-   [NPL 13] J. Am. Chem. Soc. 2015, 137, 13488-134918.-   [NPL 14] J. Am. Chem. Soc. 2012, 134, 13244-13247.-   [NPL 15] J. Am. Chem. Soc., 2016, 138, 969-974.-   [NPL 16] Tetrahedron Lett., 1998, 39, 865-868.-   [NPL 17] J. Mass. Spectrom., 1998, 33, 505-524.-   [NPL 18] J. Am. Chem. Soc., 1994, 116, 11512-11521.-   [NPL 19] J. Am. Soc. Mass Spectrom, 1995, 6, 91-101.-   [NPL 20] Int. J. Peptide Protein Res. 1996, 47, 182-189.-   [NPL 21] Int. J. Peptide Protein Res. 1988, 31, 186-191.-   [NPL 22] Molecular Biomethods Handbook, p. 515-547.-   [NPL 23] J. Mol. Model, 2013, 19, 3627-3636.-   [NPL 24] Chem. Pharm. Bull., 1981, 29, 2825-2831.-   [NPL 25] Side reactions in Peptide Synthesis, 2015, 1-31, Academic    Press.-   [NPL 26] Biochem. Biophys. Res. Commun. 1970, 40, 1173-1178.-   [NPL 27] Tetrahedron Lett., 1979, 20, 4033-4036.-   [NPL 28] Tetrahedron, 1988, 44, 805-819.-   [NPL 29] Tetrahedron Lett., 1988, 29, 1181-1184.-   [NPL 30] J. Org. Chem. 1997, 62, 3880-3889.-   [NPL 31] Org. Lett. 2018, 20, 4637-4640.-   [NPL 32] Eur. J. Org. Chem., 2012, 6204-6211-   [NPL 33] Tetrahedron Lett., 1998, 39, 7439-7442.-   [NPL 34] J. Org. Chem. 1990, 55, 2786-2797.-   [NPL 35] Bioorg. Med. Chem. Lett. 2008, 18, 3902-3905.-   [NPL 36] Tetrahedron 2004, 60, 8509-8527.-   [NPL 37] J. Org. Chem., 2016, 81, 532-544.

SUMMARY OF INVENTION Technical Problem

The present inventors subjected peptide compounds containing unnaturalamino acids to the same conditions as in the treatment with acidsrepresented by TFA that is generally used for removing protecting groupsfrom amino acids or peptides or for cleaving peptides from resins insolid-phase reactions, as generally used in conventional peptidesynthesis. As a result, the inventors found that the peptide compoundshad significant damage in the main chain, such as amide bond cleavage orrearrangement, and were not obtained efficiently with intendedsequences, leading to difficulty in manufacturing these peptidecompounds. Specifically, the inventors found that under acidicconditions the main chain is susceptible to damage at the amide bonds ofmoieties containing N-substituted (e.g. N-alkylated) amino acid forpeptide compounds containing such amino acids, or at the amide bonds ofmoieties containing aspartic acid. In addition to these problems, thepresent inventors further found many amino acid sequences that causemain-chain damage under conventional acidic conditions. An objective ofthe present invention is to provide techniques for reactions of removingprotecting groups removable under acidic conditions and reactions ofremoving resins in solid-phase reactions while suppressing sidereactions such as cleavage or rearrangement of amide bonds contained inpeptide compounds, and to provide methods of obtaining deprotectedand/or resin-removed peptide compounds in high yield and purity in asimple operation.

Solution to Problem

The present inventors conducted intensive studies mainly on reactions ofremoving protecting groups removable under acidic conditions or removingresins in solid-phase reactions while suppressing side reactions such ascleavage or rearrangement of amide bonds in peptide compounds containingN-substituted (e.g., N-alkylated) amino acids. The inventorsinvestigated techniques of reacting with a silyl compound in thepresence of an electrophilic species scavenger, and found that thetreatment of peptide compounds containing acid-labile amide bonds with asilyl compound or acid in combination with an electrophilic speciesscavenger was able to remove protecting groups and/or resins withoutinvolving main-chain damage of concern, thereby giving deprotectedand/or resin-removed target products in high yield and purity. Forreagents for such deprotection and/or resin removal, the presentinventors also found that compounds such as imidates (formula 2), amides(formula 3), ketene acetals (formula 4), ketene alkoxy hemiaminals(formula 4), enol ethers (formula 4), enol esters (formula 4), imines(formula 5), amines (formula 6), diamines (formula 7),dialkylcarbodiimides (formula 8), ureas (formula 9), or urethanes(formula 10) can be used as electrophilic species scavengers incombination with appropriate silyl compounds or acids. The presentinvention can provide target peptide compounds in higher yield andpurity without involving amide bond cleavage, more efficiently thanconventional techniques.

In a non-limiting specific embodiment, the present invention includesthe following items.

-   [1] A method of producing a peptide compound in which a protecting    group removable by a silylating agent is removed, the method    comprising the step of contacting a starting peptide compound    comprising natural amino acid residues and/or amino acid analog    residues with the silylating agent in a solvent and thereby removing    the protecting group from the starting peptide compound,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting peptide compound comprises at least one        protecting group removable by the silylating agent, and    -   wherein the starting peptide compound comprises at least one        N-substituted amino acid residue.-   [2] A method of producing a peptide compound in which a resin for    solid-phase synthesis that is removable by a silylating agent is    removed, the method comprising the step of contacting a starting    peptide compound comprising natural amino acid residues and/or amino    acid analog residues with the silylating agent in a solvent and    thereby removing the resin for solid-phase synthesis from the    starting peptide compound,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting peptide compound is linked to the removable        resin for solid-phase synthesis, and    -   wherein the starting peptide compound comprises at least one        N-substituted amino acid residue.-   [3] The method of [1] or [2], wherein the starting peptide compound    comprises at least one structure in which at least two amino acid    residues are linked to each other, wherein the structure is    represented by general formula (I) below:

-   -   wherein        -   R₁ is hydrogen, PG₁, a natural amino acid residue, or an            amino acid analog residue;        -   R₂ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl, or R₂ and R₄ or R₂ and R_(4′), together with            the nitrogen atom and carbon atom to which they are            attached, form a 3- to 7-membered heterocyclic ring            optionally substituted with hydroxy or C₁-C₄ alkoxy, wherein            R_(4′) is hydrogen when R₂ and R₄ together form the            heterocyclic ring, and R₄ is hydrogen when R₂ and R_(4′)            together form the heterocyclic ring;    -   except when R₂ and R₄, or R₂ and R_(4T) together form the        heterocyclic ring,        -   (a) R_(4′) is hydrogen, and R₄ is selected from the group            consisting of hydrogen, optionally substituted C₁-C₆ alkyl,            C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally            substituted phenyl, optionally substituted phenylmethyl,            optionally substituted phenylethyl, 2-(methylthio)ethyl,            —CH₂SPG₂, N-PG₃-indol-3-ylmethyl, 4-(PG₂O)benzyl,            PG₂-O-methyl, 1-(PG₂O)ethyl, 2-(PG₂O)ethyl, PG₂-OCO(CH₂)—,            PG₂-OCO(CH₂)₂—, PG₃N-n-butyl, —CON(R_(14A))(R_(14B)),            —CH₂—CON(R_(14A))(R_(14B)), and            —(CH₂)₂CON(R_(14A))(R_(14B)),        -   (b) R₄ and R_(4′) are independently optionally substituted            C₁-C₆ alkyl, or        -   (c) R₄ and R_(4′), together with the carbon atom to which            they are attached, form a 3- to 7-membered alicyclic ring;            -   R₅ is a single bond or —C(R_(5A))(R_(5B))—;        -   R_(5A) and R_(5B) are independently selected from the group            consisting of hydrogen, C₁-C₆ alkyl, optionally substituted            aryl, optionally substituted heteroaryl, optionally            substituted aryl-C₁-C₄ alkyl, and optionally substituted            heteroaryl-C₁-C₄ alkyl;        -   R₆ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl, or R₆ and R₇ or R₆ and R_(7′), together with            the nitrogen atom and carbon atom to which they are            attached, form a 3- to 7-membered heterocyclic ring            optionally substituted with hydroxy or C₁-C₄ alkoxy, wherein            R_(7′) is hydrogen when R₆ and R₇ together form the            heterocyclic ring, and R₇ is hydrogen when R₆ and R_(7′)            together form the heterocyclic ring;        -   except when R₆ and R₇ or R₆ and R_(7′) together form the            heterocyclic ring,        -   (a) R_(7′) is hydrogen, and R₇ is selected from the group            consisting of hydrogen, optionally substituted C₁-C₆ alkyl,            C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally            substituted phenyl, optionally substituted phenylmethyl,            optionally substituted phenylethyl, 2-(methylthio)ethyl,            —CH₂SPG₄, N-PG₅-indol-3-ylmethyl, 4-(PG₄O)benzyl,            PG₄-O-methyl, 1-(PG₄O)ethyl, 2-(PG₄O)ethyl, PG₄-OCO(CH₂)—,            PG₄-OCO(CH₂)₂—, PG₅N-n-butyl, —CON(R_(15A))(R_(11B)),            —CH₂—CON(R_(15A))(R_(15B)), and            —(CH₂)₂CON(R_(15A))(R_(15B)), or        -   (b) R₇ and R_(7′) are independently optionally substituted            C₁-C₆ alkyl, or        -   (c) R₇ and R_(7′), together with the carbon atom to which            they are attached, form a 3- to 7-membered alicyclic ring;        -   R₈ is a single bond or —C(R_(8A))(R_(8B))—;        -   R_(8A) and R_(8B) are independently selected from the group            consisting of hydrogen, C₁-C₆ alkyl, optionally substituted            aryl, optionally substituted heteroaryl, optionally            substituted aryl-C₁-C₄ alkyl, and optionally substituted            heteroaryl-C₁-C₄ alkyl;        -   R₉ is hydroxy, —O-PG₆, a natural amino acid residue, an            amino acid analog residue, —O-RES, or —NH-RES;        -   RES is a resin for solid-phase synthesis;        -   R_(14A) and R_(14B) are independently hydrogen or C₁-C₄            alkyl, or R_(14A) and R_(14B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   R_(15A) and R_(15B) are independently hydrogen or C₁-C₄            alkyl, or R_(15A) and R_(15B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   PG₁ is selected from the group consisting of Fmoc, Boc,            Alloc, Cbz, Teoc, and trifluoroacetyl;        -   PG₂ and PG₄ are independently selected from the group            consisting of hydrogen, t-Bu, trityl, methoxytrityl, cumyl,            benzyl, THP, 1-ethoxyethyl, methyl, ethyl, allyl, optionally            substituted aryl, optionally substituted aryl-C₁-C₄ alkyl,            optionally substituted heteroaryl-C1-C₄ alkyl, and            2-(trimethylsilyl)ethyl;        -   PG₃ and PG₅ are independently selected from the group            consisting of hydrogen, Fmoc, Boc, Alloc, Cbz, Teoc,            methoxycarbonyl, t-Bu, trityl, cumyl, and benzyl; and        -   PG₆ is selected from the group consisting of t-Bu, trityl,            cumyl, benzyl, methyl, ethyl, allyl, and            2-(trimethylsilyl)ethyl.

-   [4] The method of any one of [1] to [3], wherein the starting    peptide compound comprises at the C-terminus a structure in which at    least two amino acid residues are linked to each other, wherein the    structure is represented by general formula (II) below:

-   -   wherein    -   R_(1′) is a group represented by the formula (III):

-   -   * represents the point of attachment;    -   R₁ is hydrogen, PG₁, a natural amino acid residue, or an amino        acid analog residue;        -   R₂ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl, or R₂ and R₁₀, or R₂ and R_(10′), together with            the nitrogen atom and carbon atom to which they are            attached, form a 3- to 7-membered heterocyclic ring            optionally substituted with hydroxy or C₁-C₄ alkoxy, wherein            R_(10′) is hydrogen when R₂ and R₁₀ together form the            heterocyclic ring, and R₁₀ is hydrogen when R₂ and R₁₀,            together form the heterocyclic ring;        -   except when R₂ and R₁₀ or R₂ and R₁₀, together form the            heterocyclic ring,    -   (a) R_(10′) is hydrogen, and R₁₀ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₈, N-PG₉-indol-3-ylmethyl, 4-(PG₈O)benzyl, PG₈-O-methyl,        1-(PG₈O)ethyl, 2-(PG₈O)ethyl, PG₈-OCO(CH₂)—, PG₈-OCO(CH₂)₂—,        PG₉N-n-butyl, —CON(R_(16A))(R_(16B)),        —CH₂—CON(R_(16A))(R_(16B)), and —(CH₂)₂CON(R_(16A))(R_(16B)), or    -   (b) R₁₀ and R_(10′) are independently optionally substituted        C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or C₃-C₆ cycloalkyl-C₁-C₄ alkyl,        or    -   (c) R₁₀ and R_(10′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring;        -   R₁₁ is a single bond or —C(R_(11A))(R_(11B))—;        -   R_(11A) and R_(11B) are independently selected from the            group consisting of hydrogen, C₁-C₆ alkyl, optionally            substituted aryl, optionally substituted heteroaryl,            optionally substituted aryl-C₁-C₄ alkyl, and optionally            substituted heteroaryl-C₁-C₄ alkyl;        -   R₁₂ and R_(12′) are independently selected from the group            consisting of hydrogen, PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀,            —(CH₂)_(n)COO—RES, and —(CH₂)_(n)CONH—RES;        -   RES is a resin for solid-phase synthesis;        -   n is 0, 1, or 2;        -   R₆ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl;        -   R₁₃ is C₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B));        -   m is 0, 1, or 2;        -   R_(16A) and R_(16B) are independently hydrogen or C₁-C₄            alkyl, or R_(16A) and R_(16B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   R_(17A) and R_(17B) are independently hydrogen or C₁-C₄            alkyl, or R_(17A) and R_(17B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   PG₁ is independently selected from the group consisting of            Fmoc, Boc, Alloc, Cbz, Teoc, and trifluoroacetyl;        -   PG₈ is selected from the group consisting of hydrogen, t-Bu,            trityl, methoxytrityl, cumyl, benzyl, THP, 1-ethoxyethyl,            methyl, ethyl, allyl, optionally substituted aryl,            optionally substituted aryl-C₁-C₄ alkyl, optionally            substituted heteroaryl-C₁-C₄ alkyl, and            2-(trimethylsilyl)ethyl;        -   PG₉ is selected from the group consisting of hydrogen, Fmoc,            Boc, Alloc, Cbz, Teoc, methoxycarbonyl, t-Bu, trityl, cumyl,            and benzyl; and        -   PG₁₀ is selected from the group consisting of t-Bu, trityl,            cumyl, benzyl, methyl, ethyl, allyl, optionally substituted            aryl, optionally substituted aryl-C₁-C₄ alkyl, optionally            substituted heteroaryl-C₁-C₄ alkyl, and            2-(trimethylsilyl)ethyl.

-   [5] A method of producing an amide compound in which a protecting    group removable by a silylating agent is removed, the method    comprising the step of contacting a starting amide compound with the    silylating agent in a solvent and thereby removing the protecting    group from the starting amide compound,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting amide compound is represented by general        formula (II) below:

-   -   wherein    -   R_(1′) is a hydrogen atom or PG₇;    -   R₁₂ and R_(12′) are independently selected from the group        consisting of hydrogen, PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀,        —(CH₂)_(n)COO-RES, and —(CH₂)_(n)CONH-RES;    -   RES is a resin for solid-phase synthesis;    -   n is 0, 1, or 2;    -   R₆ is selected from the group consisting of hydrogen and C₁-C₆        alkyl;    -   R₁₃ is C₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B));    -   m is 0, 1, or 2;    -   R_(17A) and R_(17B) are independently hydrogen or C₁-C₄ alkyl,        or R_(17A) and R_(17B), together with the nitrogen atom to which        they are attached, form a 4- to 8-membered ring optionally        comprising one or more additional heteroatoms;    -   PG₇ is selected from the group consisting of Fmoc, Boc, Alloc,        Cbz, Teoc, and trifluoroacetyl; and    -   PG₁₀ is selected from the group consisting of t-Bu, trityl,        cumyl, benzyl, methyl, ethyl, allyl, optionally substituted        aryl, optionally substituted aryl-C₁-C₄ alkyl, optionally        substituted heteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl,        and    -   wherein the starting amide compound comprises at least one        protecting group removable by the silylating agent.

-   [6] A method of producing an amide compound in which a resin for    solid-phase synthesis is removed, the method comprising the step of    contacting a starting amide compound with a silylating agent in a    solvent and thereby removing the starting amide compound from the    resin for solid-phase synthesis,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger, and    -   wherein the starting amide compound is represented by general        formula (II) below:

-   -   wherein    -   R_(1′) is a hydrogen atom or PG₇;    -   R₁₂ and R_(12′) are independently selected from the group        consisting of hydrogen, PG₁₀-O-methyl, —(CH₂)nCOO-PG₁₀,        —(CH₂)_(n)COO-RES, and —(CH₂)_(n)CONH-RES;    -   RES is a resin for solid-phase synthesis, wherein at least one        of R₁₂ and R_(12T) is —(CH₂)_(n)COO-RES or —(CH₂)_(n)CONH-RES;    -   RES is a resin for solid-phase synthesis;    -   n is 0, 1, or 2;    -   R₆ is selected from the group consisting of hydrogen and C₁-C₆        alkyl;    -   R₁₃ is C₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B));    -   m is 0, 1, or 2;    -   R_(17A) and R_(17B) are independently hydrogen or C₁-C₄ alkyl,        or R_(17A) and R_(17B), together with the nitrogen atom to which        they are attached, form a 4- to 8-membered ring optionally        comprising one or more additional heteroatoms;    -   PG₇ is selected from the group consisting of Fmoc, Boc, Alloc,        Cbz, Teoc, and trifluoroacetyl; and    -   PG₁₀ is selected from the group consisting of t-Bu, trityl,        cumyl, benzyl, methyl, ethyl, allyl, optionally substituted        aryl, optionally substituted aryl-C₁-C₄ alkyl, optionally        substituted heteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl.

-   [7] The method of any one of [1] and [3] to [5], wherein the    removable protecting group is selected from the group consisting of    t-Bu, triphenylmethyl, 2-(trimethylsilyl)-ethyl, Boc, Teoc, Cbz,    methoxycarbonyl, tetrahydropyranyl, 1-ethoxyethyl, methoxytrityl,    and cumyl.

-   [8] The method of any one of [1] to [7], wherein the silyl compound    is represented by formula 1 below:

-   -   wherein R_(AX), R_(AY), and R_(AZ) are independently C₁-C₄ alkyl        or phenyl, and X is selected from the group consisting of —OTf,        —OClO₃, Cl, Br, and I.

-   [9] The method of [8], wherein the silyl compound is selected from    the group consisting of TMSOTf, TESOTf, TBSOTf, TIPSOTf, TBDPSOTf,    TTMSOTf, TMSCl, TMSBr, TMSOClO₃, and TMSI.

-   [10] The method of any one of [1] to [7], wherein the acid is    represented by HX, wherein X is selected from the group consisting    of —OTf, —OClO₃, Cl, Br, and I.

-   [11] The method of any one of [1] to [10], wherein the electrophilic    species scavenger is selected from the group consisting of    formulas (2) to (10) below:

-   -   wherein in formula 2,        -   R_(B) is a substituted silyl group and R_(C) is a            substituted silyl group, or        -   R_(B) and R_(C), together with the nitrogen atom and carbon            atom to which they are attached, form a 5- to 7-membered            ring; and        -   R_(D) is C₁-C₄ alkyl optionally substituted with one or more            fluorine atoms or is optionally substituted methylene,            wherein when R_(D) is optionally substituted methylene,            formula 2 is dimerized to form a compound represented by the            formula below:

-   -   wherein in formula 3,        -   R_(G) is a silyl group substituted with one or more C₁-C₄            alkyl;        -   R_(H) is hydrogen or C₁-C₄ alkyl; and        -   R_(I) is hydrogen, or C₁-C₄ alkyl optionally substituted            with one or more fluorine atoms;        -   wherein in formula 4,    -   (a-1) RJ is a substituted silyl group, RK is C1-C4 alkoxy, and        RM and RL are independently hydrogen or C1-C4 alkyl;    -   (a-2) RJ is a substituted silyl group, RM is hydrogen or C1-C4        alkyl, and RK and RL, together with the carbon atoms to which        they are attached, form a 5- to 8-membered ring comprising an        oxygen atom;    -   (b-1) RJ is a substituted silyl group, RK is C1-C4 alkyl, and RM        and RL are independently hydrogen or C1-C4 alkyl;    -   (b-2) RJ is a substituted silyl group, RM is hydrogen or C1-C4        alkyl, and RK and RL are taken together with the carbon atoms to        which they are attached, form a 5- to 8-membered ring; or    -   (c-1) RJ and RM, together with the carbon atoms to which they        are attached, form a 5- to 7-membered ring comprising an oxygen        atom, RK is hydrogen or C1-C4 alkyl, and RL is C1-C4 alkyl;    -   (c-2) RJ and RM, together with the carbon atoms to which they        are attached, form a 5- to 7-membered ring comprising an oxygen        atom, and RK and RL, together with the carbon atom to which they        are attached, form a 5- to 8-membered ring;    -   (d-1) RJ is C1-C4 alkyl and RM, RK, and RL are independently        hydrogen or C1-C4 alkyl;    -   (d-2) RJ is C1-C4 alkyl, RM is hydrogen or C1-C4 alkyl, and RK        and RL, together with the carbon atoms to which they are        attached, form a 5- to 8-membered ring;    -   (e-1) RJ is C1-C3 alkylcarbonyl and RM, RK, and RL are        independently hydrogen or C1-C4 alkyl;    -   (e-2) RJ is C1-C3 alkylcarbonyl, RM is hydrogen or C1-C4 alkyl,        and RK and RL, together with the carbon atoms to which they are        attached, form a 5- to 8-membered ring;    -   (f-1) RJ is a substituted silyl group or C1-C4 alkyl, RK is        optionally substituted di-C1-C4 alkylamino, and RM and RL are        independently hydrogen or C1-C4 alkyl; or    -   (f-2) RJ is a substituted silyl group or C1-C4 alkyl, RM is        hydrogen or C1-C4 alkyl, and RK and RL, together with the carbon        atoms to which they are attached, form a 5- to 8-membered ring        comprising a nitrogen atom, wherein the 5- to 8-membered ring is        optionally substituted with C1-C4 alkyl;        -   wherein in formula 5,        -   R_(N), R_(N′), and R_(O) are independently hydrogen or C₁-C₄            alkyl;        -   wherein in formula 6,        -   R_(P) is a substituted silyl group; and        -   R_(Q) is a substituted silyl group or C₁-C₄ alkyl and R_(R)            is hydrogen, a substituted silyl group, or C₁-C₄ alkyl, or        -   R_(Q) and R_(R), together with the nitrogen atom to which            they are attached, form a 5- to 8-membered heterocyclic ring            optionally comprising one or more additional heteroatoms;        -   wherein in formula 7,        -   X is a single bond or a carbon atom,        -   wherein when X is a single bond, R_(S) is absent, R_(UA) and            R_(R), together with the carbon atom and nitrogen atom to            which they are attached, form an optionally substituted            6-membered aromatic heterocyclic ring, and R_(UB) and R_(T),            together with the carbon atom and nitrogen atom to which            they are attached, form an optionally substituted 6-membered            aromatic heterocyclic ring, and        -   when X is a carbon atom, R_(UA) and R_(UB) are independently            C₁-C₄ alkyl and R_(R), R_(S), and R_(T), together with the            carbon atoms to which they are attached, form the structure            below:

-   -   wherein in formula 8,    -   R_(V) is C₁-C₄ alkyl or C₃-C₆ cycloalkyl;    -   wherein in formula 9,    -   R_(W) and R_(X) are independently C₁-C₄ alkyl or a substituted        silyl group; and    -   wherein in formula 10,    -   R_(Y) and R_(Z) are independently C₁-C₄ alkyl or a substituted        silyl group.

-   [12] The method of [11], wherein the electrophilic species scavenger    is selected from the group consisting of    N,O-bis(trimethylsilyl)acetamide,    N,O-bis(trimethylsilyl)trifluoroacetamide,    N-methyl-N-trimethylsilylacetamide,    N-methyl-N-trimethylsilyltrifluoroacetamide, dimethylketene methyl    trimethylsilyl acetal, isopropenyloxytrimethylsilane,    2,2,4,4-tetramethylpentanone imine, 1,1,1,3,3,3-hexamethyldisilazane    (HMDS), N-trimethylsilylmorpholine, N-trimethylsilyldiethylamine,    and N-tert-butyltrimethylsilylamine.

-   [13] The method of any one of [1] to [9] and [II] to [12], wherein    per one equivalent of the protecting group to be removed or one    equivalent of the resin to be removed, 1 to 5 equivalents of the    silyl compound and 1 to 10 equivalents of the electrophilic species    scavenger are mixed.

-   [14] The method of any one of [1] to [12], wherein per one    equivalent of the protecting group to be removed or one equivalent    of the resin to be removed, 0.1 to 0.5 equivalent of the silyl    compound or acid is mixed,    -   wherein the electrophilic species scavenger is selected from the        group consisting of N,O-bis(trimethylsilyl)acetamide,        N,O-bis(trimethylsilyl)trifluoroacetamide,        N-methyl-N-trimethylsilylacetamide,        N-methyl-N-trimethylsilyltrifluoroacetamide, dimethylketene        methyl trimethylsilyl acetal, and isopropenyloxytrimethylsilane,    -   wherein the silyl compound is selected from the group consisting        of TMSOTf, TESOTf, TBSOTf, TIPSOTf, TBDPSOTf, TTMSOTf, TMSCl,        TMSBr, and TMSOClO₃, and    -   wherein the acid is represented by HX, wherein X is selected        from the group consisting of —OTf, —OClO₃, Cl, Br, and I.

-   [15] The method of any one of [1] to [14], wherein the starting    peptide compound comprises 1 to 30 amino acid residues and is linear    or cyclic.

-   [16] The method of any one of [1] to [4] and [6] to [15], wherein    the resin for solid-phase synthesis is CTC resin, Wang resin, or    SASRIN resin.

-   [17] The method of any one of [1] to [16], wherein the method    comprises mixing the starting peptide compound with the solvent,    then with the electrophilic species scavenger, and subsequently with    the silyl compound or acid.

-   [18] The method of any one of [1] to [17], wherein the solvent is    selected from ethyl acetate, isopropyl acetate,    2-methyltetrahydrofuran, tetrahydrofuran, diethyl ether, methyl    tert-butyl ether, dichloromethane, 1,2-dichloroethane, toluene, and    acetonitrile.

-   [19] An amide compound represented by general formula (A) below or a    salt thereof:

-   -   wherein    -   R_(1′) is selected from the group consisting of hydrogen, Fmoc,        Boc, Alloc, Cbz, Teoc, and trifluoroacetyl;    -   R_(17A) and R_(17B) are both methyl, or R_(17A) and R_(17B),        together with the nitrogen atom to which they are attached, form        piperidine or morpholine; and    -   R₁₈ is hydrogen or PG₁₀, wherein PG₁₀ is selected from the group        consisting of t-Bu, trityl, cumyl, benzyl, methyl, ethyl, allyl,        optionally substituted aryl, optionally substituted aryl-C₁-C₄        alkyl, optionally substituted heteroaryl-C₁-C₄ alkyl, and        2-(trimethylsilyl)ethyl.

-   [20] The amide compound or salt thereof of [19], wherein the amide    compound is selected from the group consisting of:    -   (3-1)        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-2) allyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-3) tert-butyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-4) benzyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-5)        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-6) allyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-7) tert-butyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-8) benzyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-9)        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-10) allyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-11) tert-butyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-12) benzyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-13)        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-14) allyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-15) tert-butyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-16) benzyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-17)        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoic        acid,    -   (3-18) allyl        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,    -   (3-19) tert-butyl        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,    -   (3-20) benzyl        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,    -   (2-1)        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-2) allyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-3) tert-butyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-4) benzyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-5)        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-6) allyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-7) tert-butyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-8) benzyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-9)        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-10) allyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-11) tert-butyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-12) benzyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-13)        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-14) allyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-15) tert-butyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-16) benzyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-17)        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-18) allyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-19) tert-butyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-20) benzyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,    -   (4-1) 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoic acid,    -   (4-2) allyl 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (4-3) tert-butyl        3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (4-4) benzyl 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (4-5) 3-(methylamino)-4-morpholino-4-oxobutanoic acid,    -   (4-6) allyl 3-(methylamino)-4-morpholino-4-oxobutanoate,    -   (4-7) tert-butyl 3-(methylamino)-4-morpholino-4-oxobutanoate,    -   (4-8) benzyl 3-(methylamino)-4-morpholino-4-oxobutanoate,    -   (4-9) 4-(dimethylamino)-3-(methylamino)-4-oxobutanoic acid,    -   (4-10) allyl 4-(dimethylamino)-3-(methylamino)-4-oxobutanoate,    -   (4-11) tert-butyl        4-(dimethylamino)-3-(methylamino)-4-oxobutanoate,    -   (4-12) benzyl 4-(dimethylamino)-3-(methylamino)-4-oxobutanoate,    -   (1-1)        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-2) allyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-3) tert-butyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-4) benzyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-5)        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-6) allyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-7) tert-butyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-8) benzyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-9)        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-10) allyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-11) tert-butyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-12) benzyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-13)        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-14) allyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-15) tert-butyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-16) benzyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-17)        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-18) allyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-19) tert-butyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,        and    -   (1-20) benzyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate.

-   [21] A method of producing a peptide compound in which a protecting    group removable by a silylating agent is removed, the method    comprising the step of contacting a starting peptide compound    comprising natural amino acid residues and/or amino acid analog    residues with the silylating agent in a solvent and thereby removing    the protecting group from the starting peptide compound,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting peptide compound comprises a structure in        which two amino acid residues are linked to each other, wherein        the structure is represented by general formula (I) below:

-   -   wherein    -   R₁ is hydrogen, PG₁, a natural amino acid residue, or an amino        acid analog residue;    -   R₂ is selected from the group consisting of hydrogen and C₁-C₆        alkyl, or R₂ and R₄ or R₂ and R_(4′), together with the nitrogen        atom and carbon atom to which they are attached, form a 3- to        7-membered heterocyclic ring optionally substituted with hydroxy        or C₁-C₄ alkoxy;    -   except when R₄ and R_(4′) each, together with R₂, forms the        heterocyclic ring,    -   (a) R_(4′) is hydrogen, and R₄ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₂, N-PG₃-indol-3-ylmethyl, 4-(PG₂O)benzyl, PG₂-O-methyl,        1-(PG₂O)ethyl, 2-(PG₂O)ethyl, PG₂-OCO(CH₂)—, PG₂-OCO(CH₂)₂—,        PG₃N-n-butyl, —CON(R_(14A))(R_(14B)),        —CH₂—CON(R_(14A))(R_(14B)), and —(CH₂)₂CON(R_(14A))(R_(14B)),    -   (b) R₄ and R_(4′) are independently optionally substituted C₁-C₆        alkyl, or    -   (c) R₄ and R_(4′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring;    -   R₅ is a single bond or —C(R_(5A))(R_(5B))—;        -   R_(5A) and R_(5B) are independently selected from the group            consisting of hydrogen, C₁-C₆ alkyl, optionally substituted            aryl, optionally substituted heteroaryl, optionally            substituted aryl-C₁-C₄ alkyl, and optionally substituted            heteroaryl-C₁-C₄ alkyl;    -   R₆ is selected from the group consisting of hydrogen and C₁-C₆        alkyl, or R₆ and R₇ or R₆ and R_(7′), together with the nitrogen        atom and carbon atom to which they are attached, form a 3- to        7-membered heterocyclic ring optionally substituted with hydroxy        or C₁-C₄ alkoxy;    -   except when R₇ and R_(7′) each, together with R₆, forms the        heterocyclic ring,    -   (a) R_(7′) is hydrogen, and R₇ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₄, N-PG₅-indol-3-ylmethyl, 4-(PG₄O)benzyl, PG₄-O-methyl,        1-(PG₄O)ethyl, 2-(PG₄O)ethyl, PG₄-OCO(CH₂)—, PG₄-OCO(CH₂)₂—,        PG₅N-n-butyl, —CON(R_(15A))(R_(15B)),        —CH₂—CON(R_(15A))(R_(15B)), and —(CH₂)₂CON(R_(15A))(R_(15B)),    -   (b) R₇ and R_(7′) are independently optionally substituted C₁-C₆        alkyl, or    -   (c) R₇ and R_(7′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring;    -   R₈ is a single bond or —C(R_(8A))(R_(8B))—;    -   R_(8A) and R_(8B) are independently selected from the group        consisting of hydrogen, C₁-C₆ alkyl, optionally substituted        aryl, optionally substituted heteroaryl, optionally substituted        aryl-C₁-C₄ alkyl, and optionally substituted heteroaryl-C₁-C₄        alkyl;    -   R₉ is hydroxy, —O-PG₆, a natural amino acid residue, an amino        acid analog residue, —O-RES, or —NH-RES;    -   RES is a resin for solid-phase synthesis;    -   R_(14A) and R_(14B) are independently hydrogen or C₁-C₄ alkyl,        or R_(14A) and R_(14B), together with the nitrogen atom to which        they are attached, form a 4- to 8-membered ring optionally        comprising one or more additional heteroatoms;    -   R_(15A) and R_(15B) are independently hydrogen or C₁-C₄ alkyl,        or R_(15A) and R_(15B), together with the nitrogen atom to which        they are attached, form a 4- to 8-membered ring optionally        comprising one or more additional heteroatoms;    -   PG₁ is selected from the group consisting of Fmoc, Boc, Alloc,        Cbz, Teoc, and trifluoroacetyl;    -   PG₂ and PG₄ are independently selected from the group consisting        of hydrogen, t-Bu, trityl, methoxytrityl, cumyl, benzyl, THP,        1-ethoxyethyl, methyl, ethyl, allyl, optionally substituted        aryl, optionally substituted aryl-C₁-C₄ alkyl, optionally        substituted heteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl;    -   PG₃ and PG₅ are independently selected from the group consisting        of hydrogen, Fmoc, Boc, Alloc, Cbz, Teoc, methoxycarbonyl, t-Bu,        trityl, cumyl, and benzyl; and PG₆ is selected from the group        consisting of t-Bu, trityl, cumyl, benzyl, methyl, ethyl, allyl,        and 2-(trimethylsilyl)ethyl,        -   wherein the starting peptide compound optionally comprises            additional natural amino acid residues and/or amino acid            analog residues, and        -   wherein the starting peptide compound comprises at least one            protecting group removable by the silylating agent.

-   [22] A method of producing a peptide compound in which a resin for    solid-phase synthesis is removed, the method comprising the step of    contacting a starting peptide compound comprising natural amino acid    residues and/or amino acid analog residues with a silylating agent    in a solvent and thereby removing the starting peptide compound from    the resin for solid-phase synthesis,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting peptide compound comprises a structure in        which two amino acid residues are linked to each other, wherein        the structure is represented by general formula (I) below:

-   -   wherein        -   R₁ is hydrogen, PG₁, a natural amino acid residue, or an            amino acid analog residue;        -   R₂ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl, or R₂ and R₄ or R₂ and R_(4′), together with            the nitrogen atom and carbon atom to which they are            attached, form a 3- to 7-membered heterocyclic ring            optionally substituted with hydroxy or C₁-C₄ alkoxy;    -   except when R₄ and R_(4′) each, together with R₂, forms the        heterocyclic ring,    -   (a) R_(4′) is hydrogen, and R₄ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₂, N-PG₃-indol-3-ylmethyl, 4-(PG₂O)benzyl, PG₂-O-methyl,        1-(PG₂O)ethyl, 2-(PG₂O)ethyl, PG₂-OCO(CH₂)—, PG₂-OCO(CH₂)₂—,        PG₃N-n-butyl, —CON(R_(14A))(R_(14B)),        —CH₂—CON(R_(14A))(R_(14B)), and —(CH₂)₂CON(R_(14A))(R_(14B)),    -   (b) R₄ and R_(4′) are independently optionally substituted C₁-C₆        alkyl, or    -   (c) R₄ and R_(4′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring;        -   R₅ is a single bond or —C(R_(5A))(R_(5B))—;        -   R_(5A) and R_(5B) are independently selected from the group            consisting of hydrogen, C₁-C₆ alkyl, optionally substituted            aryl, optionally substituted heteroaryl, optionally            substituted aryl-C₁-C₄ alkyl, and optionally substituted            heteroaryl-C₁-C₄ alkyl;        -   R₆ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl, or R₆ and R₇ or R₆ and R_(7′), together with            the nitrogen atom and carbon atom to which they are            attached, form a 3- to 7-membered heterocyclic ring            optionally substituted with hydroxy or C₁-C₄ alkoxy;        -   except when R₇ and R_(7′) each, together with R₆, forms the            heterocyclic ring,    -   (a) R_(7′) is hydrogen, and R₇ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₄, N-PG₅-indol-3-ylmethyl, 4-(PG₄O)benzyl, PG₄-O-methyl,        1-(PG₄O)ethyl, 2-(PG₄O)ethyl, PG₄-OCO(CH₂)—, PG₄-OCO(CH₂)₂—,        PG₅N-n-butyl, —CON(R_(15A))(R_(15B)),        —CH₂—CON(R_(15A))(R_(15B)), and —(CH₂)₂CON(R_(15A))(R_(15B)),    -   (b) R₇ and R_(7′) are independently optionally substituted C₁-C₆        alkyl, or    -   (c) R₇ and R_(7′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring;        -   R₈ is a single bond or —C(R_(8A))(R_(8B))—;        -   R_(8A) and R_(8B) are independently selected from the group            consisting of hydrogen, C₁-C₆ alkyl, optionally substituted            aryl, optionally substituted heteroaryl, optionally            substituted aryl-C₁-C₄ alkyl, and optionally substituted            heteroaryl-C₁-C₄ alkyl;        -   R₉ is hydroxy, —O-PG₆, a natural amino acid residue, an            amino acid analog residue, —O-RES, or —NH-RES;        -   RES is a resin for solid-phase synthesis;        -   R_(14A) and R_(14B) are independently hydrogen or C₁-C₄            alkyl, or R_(14A) and R_(14B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   R_(15A) and R_(11B) are independently hydrogen or C₁-C₄            alkyl, or R_(15A) and R_(15B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   PG₁ is selected from the group consisting of Fmoc, Boc,            Alloc, Cbz, Teoc, and trifluoroacetyl;        -   PG₂ and PG₄ are independently selected from the group            consisting of hydrogen, t-Bu, trityl, methoxytrityl, cumyl,            benzyl, THP, 1-ethoxyethyl, methyl, ethyl, allyl, optionally            substituted aryl, optionally substituted aryl-C₁-C₄ alkyl,            optionally substituted heteroaryl-C₁-C₄ alkyl, and            2-(trimethylsilyl)ethyl;        -   PG₃ and PG₅ are independently selected from the group            consisting of hydrogen, Fmoc, Boc, Alloc, Cbz, Teoc,            methoxycarbonyl, t-Bu, trityl, cumyl, and benzyl; and        -   PG₆ is selected from the group consisting of t-Bu, trityl,            cumyl, benzyl, methyl, ethyl, allyl, and            2-(trimethylsilyl)ethyl,        -   wherein the starting peptide compound optionally comprises            additional natural amino acid residues and/or amino acid            analog residues, and        -   wherein the starting peptide compound comprises at least one            amino acid residue bound to the resin for solid-phase            synthesis.

-   [23] A method of producing a peptide or amide compound in which a    protecting group removable by a silylating agent is removed, the    method comprising the step of contacting a starting peptide or amide    compound comprising natural amino acid residues and/or amino acid    analog residues with the silylating agent in a solvent and thereby    removing the protecting group from the starting peptide or amide    compound,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting peptide or amide compound comprises at the        C-terminus an amino acid residue or a structure in which two        amino acid residues are linked to each other, wherein the amino        acid residue or structure is represented by general formula        (II):

-   -   wherein    -   R_(1′) is a hydrogen atom, PGP, or a group represented by        formula (III) below:

-   -   * represents the point of attachment;    -   R₁ is hydrogen, PG₁, a natural amino acid residue, or an amino        acid analog residue;    -   R₂ is selected from the group consisting of hydrogen and C₁-C₆        alkyl, or R₂ and R₁₀ or R₂ and R_(10′), together with the        nitrogen atom and carbon atom to which they are attached, form a        3- to 7-membered heterocyclic ring optionally substituted with        hydroxy or C₁-C₄ alkoxy;    -   except when R₁₀ and R_(10′) each, together with R₂, forms the        heterocyclic ring,    -   (a) R_(10′) is hydrogen, and R₁₀ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₈, N-PG₉-indol-3-ylmethyl, 4-(PG₈O)benzyl, PG₈-O-methyl,        1-(PG₈O)ethyl, 2-(PG₈O)ethyl, PG₈-OCO(CH₂)—, PG₈-OCO(CH₂)₂—,        PG₉N-n-butyl, —CON(R_(16A))(R_(16B)),        —CH₂—CON(R_(16A))(R_(16B)), and —(CH₂)₂CON(R_(16A))(R_(16B)),    -   (b) R₁₀ and R_(10′) are independently optionally substituted        C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or C₃-C₆ cycloalkyl-C₁-C₄ alkyl,        or    -   (c) R₁₀ and R_(10′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring;        -   R₁₁ is a single bond or —C(R_(11A))(R_(11B))—;        -   R_(11A) and R_(11B) are independently selected from the            group consisting of hydrogen, C₁-C₆ alkyl, optionally            substituted aryl, optionally substituted heteroaryl,            optionally substituted aryl-C₁-C₄ alkyl, and optionally            substituted heteroaryl-C₁-C₄ alkyl;        -   R₁₂ and R_(12′) are independently selected from the group            consisting of hydrogen, PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀,            —(CH₂)_(n)COO-RES, and —(CH₂)_(n)CONH-RES; RES is a resin            for solid-phase synthesis;        -   n is 0, 1, or 2;        -   R₆ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl;        -   R₁₃ is C₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B));        -   m is 0, 1, or 2;        -   R_(16A) and R_(16B) are independently hydrogen or C₁-C₄            alkyl, or R_(16A) and R_(16B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   R_(17A) and R_(17B) are independently hydrogen or C₁-C₄            alkyl, or R_(17A) and R_(17B), together with the nitrogen            atom to which they are attached, form a 4- to 8-membered            ring optionally comprising one or more additional            heteroatoms;        -   PG₁ and PG₇ are independently selected from the group            consisting of Fmoc, Boc, Alloc, Cbz, Teoc, and            trifluoroacetyl;        -   PG₈ is selected from the group consisting of hydrogen, t-Bu,            trityl, methoxytrityl, cumyl, benzyl, THP, 1-ethoxyethyl,            methyl, ethyl, allyl, optionally substituted aryl,            optionally substituted aryl-C₁-C₄ alkyl, optionally            substituted heteroaryl-C₁-C₄ alkyl, and            2-(trimethylsilyl)ethyl;        -   PG₉ is selected from the group consisting of hydrogen, Fmoc,            Boc, Alloc, Cbz, Teoc, methoxycarbonyl, t-Bu, trityl, cumyl,            and benzyl; and        -   PG₁₀ is selected from the group consisting of t-Bu, trityl,            cumyl, benzyl, methyl, ethyl, allyl, optionally substituted            aryl, optionally substituted aryl-C₁-C₄ alkyl, optionally            substituted heteroaryl-C₁-C₄ alkyl, and            2-(trimethylsilyl)ethyl,        -   wherein the starting peptide compound optionally comprises            additional natural amino acid residues and/or amino acid            analog residues, and        -   wherein the starting peptide or amide compound comprises at            least one protecting group removal by the silylating agent.

-   [24] A method of producing a peptide or amide compound in which a    resin for solid-phase synthesis is removed, the method comprising    the step of contacting a starting peptide or amide compound    comprising natural amino acid residues and/or amino acid analog    residues with a silylating agent in a solvent and thereby removing    the starting peptide or amide compound from the resin for    solid-phase synthesis,    -   wherein the silylating agent is prepared by mixing a silyl        compound or acid with an electrophilic species scavenger,    -   wherein the starting peptide or amide compound comprises at the        C-terminus an amino acid residue or a structure in which two        amino acid residues are linked to each other, wherein the amino        acid residue or structure is represented by general formula (II)        below:

-   -   wherein    -   R_(1′) is a hydrogen atom, PG₇, or a group represented by the        formula (III):

-   -   * represents the point of attachment;    -   R₁ is hydrogen, PG₁, a natural amino acid residue, or an amino        acid analog residue;    -   R₂ is selected from the group consisting of hydrogen and C₁-C₆        alkyl, or R₂ and R₁₀ or    -   R₂ and R_(10′), together with the nitrogen atom and carbon atom        to which they are attached, form a 3- to 7-membered heterocyclic        ring optionally substituted with hydroxy or C₁-C₄ alkoxy;        -   except when R₁₀ and R_(10′) each, together with R₂, forms            the heterocyclic ring,    -   (a) R_(10′) is hydrogen, and R₁₀ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₈, N-PG₉-indol-3-ylmethyl, 4-(PG₈O)benzyl, PG₈-O-methyl,        1-(PG₈O)ethyl, 2-(PG₈O)ethyl, PG₈-OCO(CH₂)—, PG₈-OCO(CH₂)₂—,        PG₉N-n-butyl, —CON(R_(16A))(R_(16B)),        —CH₂—CON(R_(16A))(R_(16B)), and —(CH₂)₂CON(R_(16A))(R_(16B)),        -   (b) R₁₀ and R_(10′) are independently optionally substituted            C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or C₃-C₆ cycloalkyl-C₁-C₄            alkyl, or        -   (c) R₁₀ and R_(10′), together with the carbon atom to which            they are attached, form a 3- to 7-membered alicyclic ring;    -   R11 is a single bond or —C(R_(11A))(R_(11B))—;    -   R11A and R11B are independently selected from the group        consisting of hydrogen, optionally substituted C1-C6 alkyl,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted aryl-C1-C4 alkyl, and optionally        substituted heteroaryl-C1-C4 alkyl;    -   R12 and R12′ are independently selected from the group        consisting of hydrogen, PG10-O-methyl, —(CH2)nCOO-PG10,        —(CH2)nCOO-RES, and —(CH2)nCONH-RES; RES is a resin for        solid-phase synthesis, wherein at least one of R12 and R12′ is        —(CH2)nCOO-RES or —(CH2)nCONH-RES;    -   RES is a resin for solid-phase synthesis;        -   n is 0, 1, or 2;        -   R₆ is selected from the group consisting of hydrogen and            C₁-C₆ alkyl;        -   R₁₃ is C₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B));        -   m is 0, 1, or 2;    -   R_(16A) and R_(16B) are independently hydrogen or C₁-C₄ alkyl,        or R_(16A) and R_(16B), together with the nitrogen atom to which        they are attached, form a 4- to 8-membered ring optionally        comprising one or more additional heteroatoms;    -   R_(17A) and R_(17B) are independently hydrogen or C₁-C₄ alkyl,        or R_(17A) and R_(17B), together with the nitrogen atom to which        they are attached, form a 4- to 8-membered ring optionally        comprising one or more additional heteroatoms;    -   PG₁ and PG₇ are independently selected from the group consisting        of Fmoc, Boc, Alloc, Cbz, Teoc, and trifluoroacetyl;    -   PG₈ is selected from the group consisting of hydrogen, t-Bu,        trityl, methoxytrityl, cumyl, benzyl, THP, 1-ethoxyethyl,        methyl, ethyl, allyl, optionally substituted aryl, optionally        substituted aryl-C1-C₄ alkyl, optionally substituted        heteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl;    -   PG₉ is selected from the group consisting of hydrogen, Fmoc,        Boc, Alloc, Cbz, Teoc, methoxycarbonyl, t-Bu, trityl, cumyl, and        benzyl; and    -   PG₁₀ is selected from the group consisting of t-Bu, trityl,        cumyl, benzyl, methyl, ethyl, allyl, optionally substituted        aryl, optionally substituted aryl-C₁-C₄ alkyl, optionally        substituted heteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl,        and    -   wherein the starting peptide compound optionally comprises        additional natural amino acid residues and/or amino acid analog        residues.

-   [25] A deprotecting agent for use in removing a protecting group,    comprising a silyl compound or acid and an electrophilic species    scavenger.

-   [26] A resin removal agent for use in cleaving a peptide compound    from a resin in a solid-phase reaction, comprising a silyl compound    or acid and an electrophilic species scavenger.

Effects of the Invention

The present invention is advantageous over known methods in that largeamounts of Bronsted acids that readily damage amide bonds are not used,the equivalents of the reagents can be reduced, the reagents used can beeasily removed, and reactions in the subsequent steps are not affected.For example, 2,6-lutidine has a high boiling point (144° C.) and istherefore rarely evaporated. Although 2,6-lutidine is a basic compoundand thus may be removed by acids, it is inefficient to remove excess2,6-lutidine under acidic conditions when the target compound is apeptide compound, because the product is a compound having a basic aminogroup. When the 2,6-lutidine used in the previous step remains in thecondensation reaction (reaction of elongating the amide bond) subsequentto the deprotection reaction, it is problematic that the condensingagent is still reacted with the remaining 2,6-lutidine, or condensationreaction still cannot be performed under desired conditions due to thepresence of a base during the condensation reaction. In contrast, forexample, the silylamines of the present invention are used moreadvantageously than 2,6-lutidine conventionally used, because thesilylamines are hydrolyzed by treatment with water after reaction andconverted to highly water-soluble compounds that can be easily removed,so that reagents derived from such silylamines can be easily removed bywashing with aqueous solutions and do not affect peptide elongation inthe subsequent step.

Among compounds used as electrophilic species scavengers such asimidates (formula 2), amides (formula 3), ketene acetals (formula 4),ketene alkoxy hemiaminals (formula 4), enol ethers (formula 4), enolesters (formula 4), imines (formula 5), amines (formula 6), diamines(formula 7), dialkylcarbodiimides (formula 8), ureas (formula 9), orurethanes (formula 10), imidates (formula 2), amides (formula 3), keteneacetals (formula 4), ketene alkoxy hemiaminals (formula 4), enol ethers(formula 4), enol esters (formula 4), or imines (formula 5) can beconverted by post-reaction mild hydrolysis treatment to compounds havinga low boiling point that can be easily removed. Therefore, excessreagents used for reaction can be removed much easier than excess2,6-lutidine. Not only amide compounds (formula 3) but also imidates areconverted by post-reaction mild hydrolysis treatment to compounds thatcan be easily removed, and thus do not affect the subsequent step.Characteristically, amines used as electrophilic species scavengers mayalso be secondary amines, in addition to tertiary amines asconventionally well-known, and such amines can achieve deprotectionwhile suppressing damage to amide bonds.

In one aspect of the present invention, N-Boc groups of peptidecompounds can be removed while suppressing damage to the main chains ofthe peptide compounds. In another aspect of the present invention, t-Buof t-Bu esters can be removed, benzyl of benzyl esters can be removed,and trityl of trityl esters can be removed at the main-chain C-terminusand in the side chains of peptide compounds while suppressing damage tothe main chains of the peptide compounds. In still another aspect of thepresent invention, peptide compounds can be removed from resins bound tothe main-chain C-terminus or the side chains of the peptide compoundswhile suppressing damage to the main chains of the peptide compounds.

DESCRIPTION OF EMBODIMENTS

The abbreviations used in the present invention are listed below.

-   -   AcOEt: Ethyl acetate    -   Alloc: Allyloxycarbonyl    -   Allyl: Allyl    -   BEP: 2-Bromo-1-ethylpyridinium tetrafluoroborate    -   Bn: Benzyl    -   Boc: tert-Butoxycarbonyl    -   BSA: N,O-Bis(trimethylsilyl)acetamide    -   BSTFA: N,O-Bis(trimethylsilyl)trifluoroacetamide    -   Bu: Butyl    -   Cbz: Benzyloxycarbonyl    -   cHx: Cyclohexyl    -   Cl-CTC resin: 2-Chlorotrityl chloride polymer resin    -   CPME: Cyclopentyl methyl ether    -   CTC: 2-Chlorotrityl chloride    -   DBU: 2,3,4,6,7,8,9,10-Octahydropyrimido[1,2-a]azepine    -   DdZ: 3,5-Dimethoxyphenylisopropoxycarbonyl    -   DIC: N,N′-Diisopropylcarbodiimide    -   DIPEA: N,N-Diisopropylethylamine    -   DMA: N,N-Dimethylacetamide    -   DME: 1,2-Dimethoxyethane    -   DMF: N,N-Dimethylformamide    -   DMSO: Dimethyl sulfoxide    -   DMT-MM:        4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium        chloride    -   EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide    -   eq.: Equivalent    -   EtOH: Ethanol    -   Et: Ethyl    -   Fmoc: 9-Fluorenylmethyloxycarbonyl    -   HATU:        1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxide hexafluorophosphate    -   HMDS: Hexamethyldisilazane    -   HOAt: 3H-1,2,3-Triazolo[4,5-b]pyridin-3-ol    -   HOBt: 1,2,3-Benzotriazol-1-ol    -   HPLC: High performance liquid chromatography    -   i-PrOAc: Isopropyl acetate    -   i-Pr: Isopropyl    -   LCMS: Liquid chromatography mass spectrometry    -   MeCN: Acetonitrile    -   Me: Methyl    -   MS: Mass spectroscopy    -   MsOH: Methanesulfonic acid    -   MSTFA: N-Methyl-N-trimethylsilyltrifluoroacetamide    -   MTBE: Methyl tert-butyl ether    -   ND: Not determined    -   NMI: 1-Methylimidazole    -   NMP: N-Methylpyrrolidone    -   oxyma: Ethyl cyano(hydroxyimino)acetate    -   Pd/C: Palladium on carbon    -   Ph: Phenyl    -   pip: Piperidinyl    -   prep.: Preparation    -   T3P: Propylphosphonic anhydride    -   TBDPS: tert-Butyldiphenylsilyl    -   TBS: tert-Butyldimethylsilyl    -   t-Bu: tert-Butyl    -   TEA: Triethylamine    -   Teoc: 2-(Trimethylsilyl)ethoxycarbonyl    -   TES: Triethylsilyl    -   TFA: Trifluoroacetic acid    -   TFE: 2,2,2-Trifluoroethanol    -   TfOH: Trifluoromethanesulfonic acid    -   Tf: Trifluoromethanesulfonyl    -   THF: Tetrahydrofuran    -   2-MeTHF: 2-Methyltetrahydrofuran    -   TIPS: Triisopropylsilyl    -   TMSOTf: Trimethylsilyl trifluoromethanesulfonate    -   TMS: Trimethylsilyl    -   Tr: Trityl    -   TTMS: Tris(trimethylsilyl)silyl    -   vol.: volume    -   Gly: Glycine    -   Ala: Alanine    -   Ser: Serine    -   Thr: Threonine    -   Val: Valine    -   Leu: Leucine    -   Ile: Isoleucine    -   Phe: Phenylalanine    -   Tyr: Tyrosine    -   Trp: Tryptophan    -   His: Histidine    -   Glu: Glutamic acid    -   Asp: Aspartic acid    -   Gln: Glutamine    -   Asn: Asparagine    -   Cys: Cysteine    -   Met: Methionine    -   Lys: Lysine    -   Arg: Arginine    -   Pro: Proline    -   MeGly: N-Me Glycine    -   MeAla: N-Me Alanine    -   MeSer: N-Me Serine    -   MeThr: N-Me Threonine    -   MeVal: N-Me Valine    -   MeLeu: N-Me Leucine    -   MeIle: N-Me Isoleucine    -   MePhe: N-Me Phenylalanine    -   MeTyr: N-Me Tyrosine    -   MeTrp: N-Me Tryptophan    -   MeHis: N-Me Histidine    -   MeGlu: N-Me Glutamic acid    -   MeAsp: N-Me Aspartic acid    -   MeGln: N-Me Glutamine    -   MeAsn: N-Me Asparagine    -   MeCys: N-Me Cysteine    -   MeMet: N-Me Methionine    -   MeLys: N-Me Lysine    -   MeArg: N-Me Arginine

Definitions of Functional Groups

The term “alkyl” as used herein refers to a monovalent group derived byremoving any one hydrogen atom from an aliphatic hydrocarbon, and coversa subset of hydrocarbyl or hydrocarbon group structures that containhydrogen and carbon atoms, but do not contain a heteroatom (which refersto an atom other than carbon and hydrogen atoms) or an unsaturatedcarbon-carbon bond in the skeleton. The “alkyl” includes linear orbranched alkyl. The alkyl is an alkyl having 1 to 20 carbon atoms(C₁-C₂₀; hereinafter, “C_(p)-C_(q)” means that it has p to q carbonatoms), examples of which include C₁-C₆ alkyl and C₁-C₄ alkyl. Specificexamples of the alkyl include methyl, ethyl, propyl, butyl, pentyl,hexyl, isopropyl, tert-butyl, and sec-butyl.

The term “cycloalkyl” as used herein refers to a saturated or partiallysaturated cyclic monovalent aliphatic hydrocarbon group, includingsingle rings, bicyclo rings, and spiro rings. The cycloalkyl may bepartially unsaturated. Preferred examples of the cycloalkyl includeC₃-C₆ cycloalkyl, which include, for example, cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl.

As used herein, the term “cycloalkylalkyl” refers to a group in whichany hydrogen atom in the above-defined “alkyl” is replaced by theabove-defined “cycloalkyl.” Preferred examples of the cycloalkylalkylinclude C₃₋₆ cycloalkyl-C₁₋₆ alkyl and C₃₋₆ cycloalkyl-C₁₋₄ alkyl.Specific examples include cyclopropylmethyl, cyclopropylethyl,cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

As used herein, the term “alkoxy” refers to an oxy group to which theabove-defined “alkyl” is bonded. Preferred examples include C₁₋₄ alkoxyand C₁₋₃ alkoxy. Specific examples of the alkoxy include methoxy,ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, sec-butoxy, andtert-butoxy.

As used herein, the term “alicyclic ring” refers to a monovalentnon-aromatic hydrocarbon ring. The alicyclic ring may have anunsaturated bond in the ring, and may be a polycyclic ring having two ormore rings. Carbon atoms constituting the ring may be oxidized to formcarbonyl. The number of atoms forming the alicyclic ring is preferably 3to 7 (3- to 7-membered alicyclic ring). Specific examples of thealicyclic ring include a cycloalkyl ring, a cycloalkenyl ring, and acycloalkynyl ring.

As used herein, the term “heterocyclic ring” refers to a non-aromaticmonovalent or divalent heterocyclic ring comprising preferably 1 to 5heteroatoms in the ring-forming atoms. The heterocyclic ring may have adouble bond and/or a triple bond in the ring, may have a carbon atom inthe ring that is oxidized to form carbonyl, and may be a single ring, afused ring, or a spiro ring. The number of the ring-forming atoms ispreferably 3 to 12 (3- to 12-membered heterocyclic ring), morepreferably 4 to 7 (4- to 7-membered heterocyclic ring), and still morepreferably 5 to 6 (5- to 6-membered heterocyclic ring).

Specific examples of the heterocyclic ring include azetidine,piperazine, pyrrolidine, piperidine, morpholine, homomorpholine,(R)-hexahydropyrrolo[1,2-a]pyrazine,(S)-hexahydropyrrolo[1,2-a]pyrazine, 3-oxopiperazine, 2-oxopyrrolidine,azetidine, 2-oxoimidazolidine, oxetane, dihydrofuran, tetrahydrofuran,dihydropyran, tetrahydropyran, tetrahydropyridine, thiomorpholine,pyrazolidine, imidazoline, oxazolidine, isoxazolidine, thiazolidine,imidazolidine, isothiazolidine, thiadiazolidine, oxazolidone,benzodioxane, benzoxazoline, dioxolane, dioxane, andtetrahydrothiopyran.

As used herein, the term “aromatic heterocyclic ring” refers to anaromatic monovalent or divalent heterocyclic ring comprising preferably1 to 5 heteroatoms in the ring-forming atoms. The aromatic heterocyclicring may be partially saturated, and may be a single ring, a fused ring(such as a bicyclic aromatic heterocyclic ring in which a monocyclicaromatic heterocyclic ring is fused with a benzene ring or a monocyclicaromatic heterocyclic ring), or a spiro ring. The number of thering-forming atoms is preferably 4 to 10 (4- to 10-membered aromaticheterocyclic ring).

Specific examples of the aromatic heterocyclic ring include furan,thiophene, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole,isoxazole, oxadiazole, thiadiazole, triazole, tetrazole, pyridine,pyrimidine, pyridazine, pyrazine, triazine, benzofuran, benzothiophene,benzothiadiazole, benzothiazole, benzoxazole, benzoxadiazole,benzimidazole, indole, isoindole, indazole, quinoline, isoquinoline,cinnoline, quinazoline, quinoxaline, indolizine, and imidazopyridine.

The term “aryl” as used herein refers to a monovalent aromatichydrocarbon ring, preferred examples of which include C₆-C₁₀ aryl.Specific examples of the aryl include phenyl and naphthyl (e.g.,1-naphthyl or 2-naphthyl).

The term “heteroaryl” as used herein refers to a monovalent aromaticheterocyclic group comprising preferably 1 to 5 heteroatoms in thering-forming atoms“ ”. The heteroaryl may be partially saturated, andmay be a single ring or fused rings (such as bicyclic heteroaryl inwhich heteroaryl is fused with benzene ring or monocyclic heteroarylring). The number of the ring-forming atoms is preferably 5 to 10 (5- to10-membered heteroaryl).

Specific examples of the heteroaryl include furyl, thienyl, pyrrolyl,imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl,oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl,pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl,benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl,benzimidazolyl, indolyl, isoindolyl, azaindolyl, indazolyl, quinolyl,isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl,indolizinyl, and imidazopyridyl.

As used herein, the term “heterocyclyl” refers to a non-aromaticmonovalent heterocyclic group comprising preferably 1 to 5 heteroatomsin the ring-forming atoms. The heterocyclyl may have a double or triplebond in the ring, may have a carbon atom oxidized to form carbonyl, andmay be a single ring or a fused ring. The number of the ring-formingatoms is preferably 3 to 10 (3- to 10-membered heterocyclyl).

Specific examples of the heterocyclyl include oxetanyl, dihydrofuryl,tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl,morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, thiadiazolidinyl,azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, anddioxanyl.

As used herein, the term “arylalkyl” refers to a group in which anyhydrogen atom in the above-defined “alkyl” is replaced by theabove-defined “aryl.” Preferred examples of the arylalkyl include C6-10aryl-C1-4 alkyl and C6-10 aryl-C1-3 alkyl. Specific examples includebenzyl, phenylmethyl, phenethyl (phenylethyl), and naphthylmethyl.

As used herein, the term “heteroarylalkyl” refers to a group in whichany hydrogen atom in the above-defined “alkyl” is replaced by theabove-defined “heteroaryl.” Preferred examples of the heteroarylalkylinclude 5- to 10-membered heteroaryl-C1-3 alkyl. Specific examplesinclude pyrrolylmethyl, imidazolylmethyl, thienylmethyl, pyridylmethyl,pyrimidylmethyl, quinolylmethyl, and pyridylethyl.

As used herein, the term “substituted silyl” refers to a silyl groupsubstituted with 1 to 3 substituents. The substituents may be the sameor different. Such substituents are preferably C1-C6 alkyl, aryl, andtri-C1-C6 alkylsilyl. Specific examples include trimethylsilyl,triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, and tris(trimethylsilyl)silyl.

When the modifier “optionally substituted” is provided herein, examplesof such substituents include alkyl, alkoxy, alkenyl, alkenyloxy,alkynyl, alkynyloxy, cycloalkyl, aryl, heteroaryl, heterocyclyl,arylalkyl, heteroarylalkyl, substituted silyl, halogen atoms, nitro,amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl,and formyl.

As used herein, the term “deprotection/removal of a protection group”refers to converting a protected functional group back to an originalfunctional group by removing the protecting group.

As used herein, the term “resin removal/removal of a resin” refers tocleaving a peptide compound from a resin for solid-phase synthesis thatis bound to the peptide compound. The resin for solid-phase synthesis ispreferably bound to the C-terminal amino acid residue of the startingpeptide compound.

In one embodiment, the present invention provides methods of producingpeptide pharmaceuticals comprising amino acid analogs useful for peptidepharmaceuticals. In another embodiment, the present invention providesmethods of producing peptides comprising high-quality amino acid analogsfor supplying pharmaceutical drug substances. In still anotherembodiment, the present invention provides novel amide compounds usefulfor producing peptide compounds.

Deprotection/Resin Removal Methods

In some aspects, the present invention relates to a method of producinga peptide compound in which a protecting group removable by a silylatingagent is removed, the method comprising the step of contacting astarting peptide compound comprising natural amino acid residues and/oramino acid analog residues with the silylating agent in a solvent andthereby removing the protecting group from the starting peptidecompound.

In some aspects, the present invention relates to a method of producinga peptide compound in which a resin for solid-phase synthesis isremoved, the method comprising the step of contacting a starting peptidecompound comprising natural amino acid residues and/or amino acid analogresidues with a silylating agent in a solvent and thereby removing thestarting peptide compound from the resin for solid-phase synthesis.

As used herein, the term “starting peptide compound” refers to a“peptide compound” which is a starting material to be subjected to thedeprotection reaction and/or resin removal reaction of the presentinvention. The starting peptide compound preferably comprises at leastone N-substituted amino acid residue.

“Peptide compounds” in the present invention include linear or cyclicpeptide compounds comprising natural amino acid residues and/or aminoacid analog residues. Cyclic peptide compounds are synonymous with“peptide compounds having a cyclic moiety.”

As used in the present invention, “linear peptide compounds” are notparticularly limited as long as they are peptide compounds that areformed by natural amino acids or amino acid analogs forming amide orester bonds and that do not have a cyclic moiety. Such a linear peptidecompound can be formed by a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 20, 25, or 30 natural amino acids or amino acid analogs.The number of amino acids constituting the linear peptide compoundpreferably ranges from 1 to 30, from 6 to 20, from 7 to 19, from 7 to18, from 7 to 17, from 7 to 16, from 7 to 15, from 8 to 14, or from 9 to13.

As used in the present invention, “peptide compounds having a cyclicmoiety” are not particularly limited as long as they are peptidecompounds that are formed by natural amino acids or amino acid analogsforming amide or ester bonds and that have a cyclic moiety. Such cyclicmoieties are preferably formed through covalent bonds such as amidebonds, carbon-carbon bonds, S—S bonds, thioether bonds, and triazolebonds (WO2013/100132, WO2012/026566, WO2012/033154, WO2012/074130,WO2015/030014, Comb Chem High Throughput Screen. 2010; 13:75-87, NatureChem. Bio. 2009, 5, 502, Nat Chem Biol. 2009, 5, 888-90, BioconjugateChem., 2007, 18, 469-476, ChemBioChem, 2009, 10, 787-798, ChemicalCommunications (Cambridge, United Kingdom) (2011), 47(36), 9946-9958).Compounds obtained by further chemically modifying such compounds arealso included in the peptide compounds of the present invention. Thepeptide compounds of the present invention having a cyclic moiety mayalso have a linear moiety. The number of amide or ester bonds (thenumber or length of natural amino acids or amino acid analogs) is notparticularly limited, but when the peptide compound has a linear moiety,the cyclic and linear moieties combined preferably have 30 residues orless. The total number of amino acids is more preferably 9 or more inorder to achieve high metabolic stability. In addition to above, thecyclic moiety is preferably formed by 5 to 12, 6 to 12, or 7 to 12,still more preferably 7 to 11 or 8 to 11, and particularly preferably 9to 11 (10 or 11), natural amino acids and amino acid analogs. The linearmoiety preferably has 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4, andmore preferably 0 to 3, amino acids and amino acid analogs. The totalnumber of natural amino acids and amino acid analogs is preferably 1 to30, 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, or 9to 13.

Although there are no particular limitations on the types of naturalamino acid residues and amino acid analog residues forming the cyclicmoiety of the peptide compound of the present invention having a cyclicmoiety, the cyclic moiety is preferably formed by natural amino acidresidues and amino acid analog residues having a functional group withhigh metabolic stability. The method of cyclizing the peptide compoundof the present invention having a cyclic moiety is not particularlylimited as long as the method can form such a cyclic moiety. Examples ofthe cyclization method include amide bond formation from carboxylicacids and amines; and carbon-carbon bond formation reactions usingtransition metals as catalysts such as Suzuki reaction, Heck reaction,and Sonogashira reaction. Accordingly, the peptide compound of thepresent invention contains at least one set of functional groupsallowing such bond formation reactions before cyclization. Inparticular, in terms of metabolic stability, it preferably containsfunctional groups that form an amide bond by bond formation reaction.

Preferably, the cyclic moiety formed does not include a bond thatcontains a heteroatom which may be readily oxidized and that hindersmetabolic stability, for example. Examples of bonds produced bycyclization include amide bonds formed by active esters and amines andbonds produced by Heck reaction products from carbon-carbon double bondsand aryl halides.

The “peptide compound” of the present invention may be a linear orcyclic peptide comprising at least one N-substituted amino acid(preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or30, particularly preferably 5, 6, or 7, N-substituted amino acids) andat least one N-unsubstituted amino acid, under or independently of theabove-described conditions of the total number of natural amino acidsand amino acid analogs. The number of N-substituted amino acids ispreferably within the range of 1 to 30, 6 to 20, 7 to 19, 7 to 18, 7 to17, 7 to 16, 7 to 15, 8 to 14, or 9 to 13.

In some aspects, the starting peptide compound comprises 1 to 30 aminoacid residues and is linear or cyclic. Amino acids contained in thestarting peptide compound may be either “natural amino acids” or “aminoacid analogs.” “Amino acids”, “natural amino acids”, and “amino acidanalogs” may be referred to herein as “amino acid residues,” “naturalamino acid residues,” and “amino acid analog residues,” respectively.The starting peptide compound may be formed by secondary amides,tertiary amides, or a mixture of secondary and tertiary amides.

The term “natural amino acid” refers to Gly, Ala, Ser, Thr, Val, Leu,Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, or Pro.

“Amino acid analogs” are not particularly limited, and include β-aminoacids, γ-amino acids, D-amino acids, N-substituted amino acids,α,α-disubstituted amino acids, hydroxycarboxylic acids, and unnaturalamino acids (amino acids whose side chains are different from those ofnatural amino acids: for example, unnatural α-amino acids, β-aminoacids, and γ-amino acids). An α-amino acid may be a D-amino acid, or anα,α-dialkylamino acid. In a similar manner to an α-amino acid, a β-aminoacid and a γ-amino acid are also allowed to have any configuration.Examples of N-substituted amino acids include amino acids of which theamino groups are substituted with any substituents. Examples of suchsubstituents include, but are not particularly limited to, an alkylgroup, an aryl group, and an aralkyl group. N-substituted amino acidsinclude N-alkylamino acids, N-arylamino acids, and N-methylamino acids.There is no particular limitation on the selection of amino acid sidechain, but in addition to a hydrogen atom, it can be freely selectedfrom, for example, an alkyl group, an alkenyl group, an alkynyl group,an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkylgroup. One or two non-adjacent methylene groups in such a group areoptionally substituted with an oxygen atom, a carbonyl group (—CO—), ora sulfonyl group (—SO2-). Each group may have one, two or moresubstituent(s). For example, the substituents are freely selected fromany functional groups including a halogen atom, an N atom, an O atom, anS atom, a B atom, an Si atom, or a P atom (i.e., an optionallysubstituted alkyl group, alkenyl group, alkynyl group, aryl group,heteroaryl group, aralkyl group, and cycloalkyl group).

“Natural amino acids” and “amino acid analogs” as used herein whichconstitute a peptide compound include all isotopes corresponding to eachamino acid. The isotope of the “natural amino acid” or “amino acidanalog” refers to one having at least one atom replaced with an atom ofthe same atomic number (number of protons) and different mass number(total number of protons and neutrons). Examples of isotopes containedin the “natural amino acid” or “amino acid analog” constituting thepeptide compounds of the present invention include a hydrogen atom, acarbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, asulfur atom, a fluorine atom, and a chlorine atom, which respectivelyinclude ²H and ³H; ¹³C and ¹⁴C; ¹⁵N; ¹⁷O and ¹¹O; ³²P; ³⁵S; ¹⁸F; and³⁶Cl.

Examples of substituents containing a halogen atom as used hereininclude a halogen-substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, or aralkyl. More specific examples include fluoroalkyl,difluoroalkyl, and trifluoroalkyl.

Substituents containing an O atom include groups such as hydroxy (—OH),oxy (—OR), carbonyl (—C═O—R), carboxy (—CO2H), oxycarbonyl (—C═O—OR),carbonyloxy (—O—C═O—R), thiocarbonyl (—C═O—SR), carbonylthio (—S—C═O—R),aminocarbonyl (—C═O—NHR), carbonylamino (—NH—C═O—R), oxycarbonylamino(—NH—C═O—OR), sulfonylamino (—NH—SO2-R), aminosulfonyl (—SO2-NHR),sulfamoylamino (—NH—SO2-NHR), thiocarboxyl (—C(═O)—SH), andcarboxylcarbonyl (—C(═O)—CO2H).

Examples of oxy (—OR) include alkoxy, cycloalkoxy, alkenyloxy,alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy. The alkoxy ispreferably C1-C4 alkoxy and C1-C2 alkoxy, and particularly preferablymethoxy or ethoxy.

Examples of carbonyl (—C═O—R) include formyl (—C═O—H), alkylcarbonyl,cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl,heteroarylcarbonyl, and aralkylcarbonyl.

Examples of oxycarbonyl (—C═O—OR) include alkyloxycarbonyl,cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl.

Examples of carbonyloxy (—O—C═O—R) include alkylcarbonyloxy,cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.

Examples of thiocarbonyl (—C═O—SR) include alkylthiocarbonyl,cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl,arylthiocarbonyl, heteroarylthiocarbonyl, and aralkylthiocarbonyl.

Examples of carbonylthio (—S—C═O—R) include alkylcarbonylthio,cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio,arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio.

Examples of aminocarbonyl (—C═O—NHR) include alkylaminocarbonyl(examples of which include C1-C6 or C1-C4 alkylaminocarbonyl, inparticular, ethylaminocarbonyl and methylaminocarbonyl),cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl,arylaminocarbonyl, heteroarylaminocarbonyl, and aralkylaminocarbonyl.Additional examples include compounds in which the H atom bonded to theN atom in —C═O—NHR is further replaced with alkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, or aralkyl.

Examples of carbonylamino (—NH—C═O—R) include alkylcarbonylamino,cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino,arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino.Additional examples include compounds in which the H atom bonded to theN atom in —NH—C═O—R is further replaced with alkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, or aralkyl.

Examples of oxycarbonylamino (—NH—C═O—OR) include alkoxycarbonylamino,cycloalkoxycarbonylamino, alkenyloxycarbonylamino,alkynyloxycarbonylamino, aryloxycarbonylamino,heteroaryloxycarbonylamino, and aralkyloxycarbonylamino. Additionalexamples include compounds in which the H atom bonded to the N atom in—NH—C═O—OR is further replaced with alkyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl, or aralkyl.

Examples of sulfonylamino (—NH—SO2-R) include alkylsulfonylamino,cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino,arylsulfonylamino, heteroarylsulfonylamino, and aralkylsulfonylamino.Additional examples include compounds in which the H atom attached tothe N atom in —NH—SO2-R is further replaced with alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

Examples of aminosulfonyl (—SO2-NHR) include alkylaminosulfonyl,cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl,arylaminosulfonyl, heteroarylaminosulfonyl, and aralkylaminosulfonyl.Additional examples include compounds in which the H atom attached tothe N atom in —SO2-NHR is further replaced with alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

Examples of sulfamoylamino (—NH—SO2-NHR) include alkylsulfamoylamino,cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino,arylsulfamoylamino, heteroarylsulfamoylamino, and aralkylsulfamoylamino.The two H atoms bonded to the N atoms in —NH—SO2-NHR may be furtherreplaced with substituents independently selected from the groupconsisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, andaralkyl, and these two substituents may form a ring.

Substituents containing an S atom include groups such as thiol (—SH),thio (—S—R), sulfinyl (—S═O—R), sulfonyl (—SO2-R), and sulfo (—SO3H).

Examples of thio (—S—R) include alkylthio, cycloalkylthio, alkenylthio,alkynylthio, arylthio, heteroarylthio, and aralkylthio.

Examples of sulfonyl (—SO2-R) include alkylsulfonyl, cycloalkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, andaralkylsulfonyl.

Substituents containing an N atom include groups such as azido (—N3,also called “azido group”), cyano (—CN), primary amino (—NH2), secondaryamino (—NH—R; also called monosubstituted amino), tertiary amino(—NR(R′); also called disubstituted amino), amidino (—C(═NH)—NH2),substituted amidino (—C(═NR)—NR′R″), guanidino (—NH—C(═NH)—NH2),substituted guanidino (—NR—C(═NR″′)—NR′R″), aminocarbonylamino(—NR—CO—NR′R″), pyridyl, piperidino, morpholino, and azetidinyl.

Examples of secondary amino (—NH—R; monosubstituted amino) includealkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino,heteroarylamino, and aralkylamino.

Examples of tertiary amino (—NR(R′); disubstituted amino) include aminogroups having any two substituents each independently selected fromalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, suchas alkyl(aralkyl)amino, where any two such substituents may form a ring.Specific examples include dialkylamino, in particular, C1-C6dialkylamino, C1-C4 dialkylamino, dimethylamino, and diethylamino. Theterm “Cp-Cq dialkylamino group” as used herein refers to an amino groupsubstituted with two Cp-Cq alkyl groups, where the two Cp-Cq alkylgroups may be the same or different.

Examples of substituted amidino (—C(═NR)—NR′R″) include groups in whichthree substituents R, R′, and R″ on the N atom are each independentlyselected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, andaralkyl, such as alkyl(aralkyl)(aryl)amidino.

Examples of substituted guanidino (—NR—C(═NR″′)—NR′R″) include groups inwhich R, R′, R″, and R″′ are each independently selected from alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groupsin which these substituents form a ring.

Examples of aminocarbonylamino (—NR—CO—NR′R″) include groups in which R,R′, and R″ are each independently selected from a hydrogen atom, alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groupsin which these substituents form a ring.

In some aspects, the starting peptide compound to be subjected todeprotection and/or resin removal comprises at least one N-substitutedamino acid residue. Examples of the number of N-substituted amino acidresidues contained in the starting peptide compound include 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30. The number ofN-substituted amino acid residues is preferably within the range of 6 to20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, or 9 to 13.Two or more N-substituted amino acid residues may be linked to eachother in the starting peptide compound.

In some aspects, half or more of the amino acids constituting thepeptide compound of the present invention (for example, n or more aminoacids (where n is an integer) when the peptide compound is constitutedby 2n amino acids, or n+1 or more amino acids when the peptide compoundis constituted by 2n+1 amino acids) are preferably N-substituted aminoacids.

As used herein, the term “N-substitution” in N-substituted amino acidsrefers to, but is not limited to, replacement of a hydrogen atomattached to an N atom with a methyl group, an ethyl group, a propylgroup, a butyl group, or a hexyl group. Preferred N-substituted aminoacids include amino acids in which amino groups contained in naturalamino acids are N-methylated, N-ethylated, N-propylated, N-butylated,and N-pentylated. Such amino acids are called N-methylamino acid,N-ethylamino acid, N-propylamino acid, N-butylamino acid, andN-pentylamino acid.

When the starting peptide compound comprises at least one N-substitutedamino acid residue (for example, when the starting peptide compoundcomprises one or more partial structures in which an N-substituted aminoacid residue is linked to the adjacent amino acid residue, morespecifically, one or more partial structures each comprising at leasttwo amino acid residues, which structure is represented by generalformulas (I) and/or (II)), conventional deprotection/resin removalmethods easily cause main-chain damage such as amide bond cleavage orpeptide main-chain rearrangement. Even in case of such starting peptidecompounds, use of the methods of the present invention is able to removeprotecting groups of interest efficiently in high yield and puritywithout involving main-chain damage, and to cleave peptide compoundsfrom resins in solid-phase reactions efficiently in high yield andpurity.

In some embodiments, the starting peptide compound of the presentinvention may not comprise N-substituted amino acid residues or apartial structure in which an N-substituted amino acid residue is linkedto the adjacent amino acid residue.

In some aspects, the starting peptide compound to be subjected todeprotection and/or resin removal comprises at least one partialstructure in which at least two amino acid residues are linked to eachother, wherein the structure is represented by general formula (I)below.

In formula (I), R₁ is hydrogen, PG₁, a natural amino acid residue, or anamino acid analog residue.

When the structure represented by formula (I) is at the N-terminus ofthe starting peptide compound, R₁ is preferably hydrogen or PG₁. On theother hand, when the structure represented by formula (I) is at aposition other than the N-terminus of the starting peptide compound, R₁is preferably a natural amino acid residue or an amino acid analogresidue.

In formula (I), R₂ is selected from the group consisting of hydrogen andC₁-C₆ alkyl, or R₂ and R₄ or R₂ and R_(4′), together with the nitrogenatom and carbon atom to which they are attached, form a 3- to 7-memberedheterocyclic ring optionally substituted with hydroxy or C₁-C₄ alkoxy.

In some aspects, when R₂ is C₁-C₆ alkyl, the C₁-C₆ alkyl is preferablymethyl, ethyl, propyl, butyl, or pentyl.

In some aspects, when R₂ and R₄ or R₂ and R_(4′), together with thenitrogen atom and carbon atom to which they are attached, form a 3- to7-membered heterocyclic ring optionally substituted with hydroxy orC₁-C₄ alkoxy, the 3- to 7-membered heterocyclic ring formed ispreferably an azetidine ring, a pyrrolidine ring, or a piperidine ring.R_(4′) is hydrogen when R₂ and R₄ together form the heterocyclic ring,and R₄ is hydrogen when R₂ and R_(4′) together form the heterocyclicring.

In formula (I), except when R₂ and R₄ or R₂ and R₄, together form theheterocyclic ring,

-   -   (a) R_(4′) is hydrogen, and R₄ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₂, N-PG₃-indol-3-ylmethyl, 4-(PG₂O)benzyl, PG₂-O-methyl        (i.e., —CH₂—O-PG₂), 1-(PG₂O)ethyl, 2-(PG₂O)ethyl, PG₂-OCO(CH₂)—,        PG₂-OCO(CH₂)₂—, PG₃N-n-butyl, —CON(R_(14A))(R_(14B)),        —CH₂—CON(R_(14A))(R_(14B)), and —(CH₂)₂CON(R_(14A))(R_(14B)),    -   (b) R₄ and R_(4′) are independently optionally substituted C₁-C₆        alkyl, or    -   (c) R₄ and R_(4′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring.

Combinations of R₄ and R_(4′) are preferably a hydrogen atom and ahydrogen atom, methyl and a hydrogen atom, ethyl and a hydrogen atom,isopropyl and a hydrogen atom, isobutyl and a hydrogen atom, cyclopropyland a hydrogen atom, cyclopropylmethyl and a hydrogen atom, cyclopentyland a hydrogen atom, cyclohexyl and a hydrogen atom, optionallysubstituted phenyl and a hydrogen atom, optionally substitutedphenylmethyl and a hydrogen atom, optionally substituted phenylethyl anda hydrogen atom, 2-(methylthio)ethyl and a hydrogen atom, —CH₂SPG₂ and ahydrogen atom, N-PG₃-indol-3-ylmethyl and a hydrogen atom,4-(PG₂O)benzyl and a hydrogen atom, PG₂-O-methyl and a hydrogen atom,1-(PG₂O)ethyl and a hydrogen atom, 2-(PG₂O)ethyl and a hydrogen atom,PG₂-OCO(CH₂)— and a hydrogen atom, PG₂-OCO(CH₂)₂— and a hydrogen atom,PG₃N-n-butyl and a hydrogen atom, —CON(R_(14A))(R_(14B)) and a hydrogenatom, —CH₂—CON(R_(14A))(R_(14B)) and a hydrogen atom,—(CH₂)₂CON(R_(14A))(R_(14B)) and a hydrogen atom, methyl and methyl, andmethyl and ethyl. When R₄ and R_(4′), together with the carbon atom towhich they are attached, form a 3- to 7-membered alicyclic ring, thealicyclic ring is preferably a cyclopropyl ring, a cyclobutyl ring, acyclopentyl ring, or a cyclohexyl ring.

In formula (I), R₅ is a single bond or —C(R_(5A))(R_(5B))—; and R_(5A)and R_(5B) are independently selected from the group consisting ofhydrogen, C1-C₆ alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryl-C1-C₄ alkyl, andoptionally substituted heteroaryl-C₁-C₄ alkyl.

R₅ is preferably a single bond or —C(R_(5A))(R_(5B))— where thecombinations of R_(5A) and R_(5B) is a hydrogen atom and a hydrogenatom, methyl and a hydrogen atom, ethyl and a hydrogen atom, isopropyland a hydrogen atom, isobutyl and a hydrogen atom, cyclopropyl and ahydrogen atom, cyclopropylmethyl and a hydrogen atom, optionallysubstituted phenyl and a hydrogen atom, optionally substitutedphenylmethyl and a hydrogen atom, optionally substituted phenylethyl anda hydrogen atom, methyl and methyl, or methyl and ethyl.

In formula (I), R₆ is selected from the group consisting of hydrogen andC₁-C₆ alkyl, or R₆ and R₇ or R₆ and R_(7′), together with the nitrogenatom and carbon atom to which they are attached, form a 3- to 7-memberedheterocyclic ring optionally substituted with hydroxy or C₁-C₄ alkoxy.

In some aspects, when R₆ is C₁-C₆ alkyl, the C₁-C₆ alkyl is preferablymethyl, ethyl, propyl, butyl, or pentyl.

In some aspects, when R₆ and R₇ or R₆ and R_(7′), together with thenitrogen atom and carbon atom to which they are attached, form a 3- to7-membered heterocyclic ring optionally substituted with hydroxy orC1-C4 alkoxy, the 3- to 7-membered heterocyclic ring formed ispreferably an azetidine ring, a pyrrolidine ring, or a piperidine ring.R7′ is hydrogen when R6 and R7 together form the heterocyclic ring, andR7 is hydrogen when R6 and R7′ together form the heterocyclic ring.

In formula (I), preferably, either or both of R2 and R6 are other thanhydrogen, and more preferably, either or both of R2 and R6 are C1-C6alkyl.

In formula (I), except when R₆ and R₇, or R₆ and R_(7′) together formthe heterocyclic ring,

-   -   (a) R_(7′) is hydrogen, and R₇ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₄, N-PG₅-indol-3-ylmethyl, 4-(PG₄O)benzyl, PG₄-O-methyl        (i.e., —CH₂—O-PG₄), 1-(PG₄O)ethyl, 2-(PG₄O)ethyl, PG₄-OCO(CH₂)—,        PG₄-OCO(CH₂)₂—, PG₅N-n-butyl, —CON(R_(15A))(R_(15B)),        —CH₂—CON(R_(15A))(R₁₅), and —(CH₂)₂CON(R_(15A))(R_(15B)),    -   (b) R₇ and R_(7′) are independently optionally substituted C₁-C₆        alkyl, or    -   (c) R₇ and R_(7′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring.

Combinations of R₇ and R_(7′) are preferably a hydrogen atom and ahydrogen atom, methyl and a hydrogen atom, ethyl and a hydrogen atom,isopropyl and a hydrogen atom, isobutyl and a hydrogen atom, cyclopropyland a hydrogen atom, cyclopropylmethyl and a hydrogen atom, cyclopentyland a hydrogen atom, cyclohexyl and a hydrogen atom, optionallysubstituted phenyl and a hydrogen atom, optionally substitutedphenylmethyl and a hydrogen atom, optionally substituted phenylethyl anda hydrogen atom, 2-(methylthio)ethyl and a hydrogen atom, —CH₂SPG₄ and ahydrogen atom, N-PG₅-indol-3-ylmethyl and a hydrogen atom,4-(PG₄O)benzyl and a hydrogen atom, PG₄-O-methyl and a hydrogen atom,1-(PG₄O)ethyl and a hydrogen atom, 2-(PG₄O)ethyl and a hydrogen atom,PG₄-OCO(CH₂)— and a hydrogen atom, PG₄-OCO(CH₂)₂— and a hydrogen atom,PG₅N-n-butyl and a hydrogen atom, —CON(R_(15A))(R_(15B)) and a hydrogenatom, —CH₂—CON(R_(15A))(R_(15B)) and a hydrogen atom,—(CH₂)₂CON(R_(15A))(R_(15B)) and a hydrogen atom, methyl and methyl, andmethyl and ethyl. When R₇ and R_(7′), together with the carbon atom towhich they are attached, form a 3- to 7-membered alicyclic ring, thealicyclic ring is preferably a cyclopropyl ring, a cyclobutyl ring, acyclopentyl ring, or a cyclohexyl ring.

In formula (I), R₈ is a single bond or —C(R_(8A))(R_(8B))—; and R_(8A)and R_(8B) are independently selected from the group consisting ofhydrogen, C₁-C₆ alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryl-C₁-C₄ alkyl, andoptionally substituted heteroaryl-C₁-C₄ alkyl.

R₈ is preferably a single bond or —C(R_(8A))(R_(8B))— where thecombination of R_(8A) and R_(8B) is a hydrogen atom and a hydrogen atom,methyl and a hydrogen atom, ethyl and a hydrogen atom, isopropyl and ahydrogen atom, isobutyl and a hydrogen atom, cyclopropyl and a hydrogenatom, cyclopropylmethyl and a hydrogen atom, optionally substitutedphenyl and a hydrogen atom, optionally substituted phenylmethyl and ahydrogen atom, optionally substituted phenylethyl and a hydrogen atom,methyl and methyl, or methyl and ethyl.

In formula (I), R₉ is hydroxy, —O-PG₆, a natural amino acid residue, anamino acid analog residue, —O-RES, or —NH-RES, where RES is a resin forsolid-phase synthesis.

When the structure represented by formula (I) is at the C-terminus ofthe starting peptide compound, R₉ is preferably hydroxy, —O-PG₆, —O-RES,or —NH-RES. On the other hand, when the structure represented by formula(I) is at a position other than the C-terminus of the starting peptidecompound, R₉ is preferably a natural amino acid residue or an amino acidanalog residue.

In formula (I), R_(14A) and R_(14B) are independently hydrogen or C₁-C₄alkyl, or R_(14A) and R_(14B), together with the nitrogen atom to whichthey are attached, form a 4- to 8-membered ring optionally comprisingone or more additional heteroatoms. When R_(14A) and/or R_(14B) areC₁-C₄ alkyl, the C₁-C₄ alkyl is preferably methyl, ethyl, or propyl.When R_(14A) and R_(14B), together with the nitrogen atom to which theyare attached, form a 4- to 8-membered ring optionally comprising one ormore additional heteroatoms, the 4- to 8-membered ring is preferably anazetidine ring, a pyrrolidine ring, a piperidine ring, a piperazinering, or a morpholine ring.

In formula (I), R_(15A) and R_(15B) are independently hydrogen or C₁-C₄alkyl, or R_(15A) and R_(15B), together with the nitrogen atom to whichthey are attached, form a 4- to 8-membered ring optionally comprisingone or more additional heteroatoms. When R_(15A) and/or R_(15B) areC₁-C₄ alkyl, the C₁-C₄ alkyl is preferably methyl, ethyl, or propyl.When R_(15A) and R_(15B), together with the nitrogen atom to which theyare attached, form a 4- to 8-membered ring optionally comprising one ormore additional heteroatoms, then the 4- to 8-membered ring ispreferably an azetidine ring, a pyrrolidine ring, a piperidine ring, apiperazine ring, or a morpholine ring.

In formula (I), PG₁ is selected from the group consisting of Fmoc, Boc,Alloc, Cbz, Teoc, and trifluoroacetyl.

PG₁ is preferably Fmoc, Boc, or Cbz.

In formula (I), PG₂ and PG₄ are independently selected from the groupconsisting of hydrogen, t-Bu, trityl, methoxytrityl, cumyl, benzyl, THP,1-ethoxyethyl, methyl, ethyl, allyl, optionally substituted aryl,optionally substituted aryl-C₁-C₄ alkyl, optionally substitutedheteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl.

PG₂ and PG₄ are preferably methyl, allyl, t-Bu, trityl, methoxytrityl,cumyl, THP, optionally substituted aryl, optionally substitutedaryl-C₁-C₄ alkyl, or optionally substituted heteroaryl-C₁-C₄ alkyl.

In formula (I), PG₃ and PG₅ are independently selected from the groupconsisting of hydrogen, Fmoc, Boc, Alloc, Cbz, Teoc, methoxycarbonyl,t-Bu, trityl, cumyl, and benzyl.

PG₃ and PG₅ are preferably Fmoc, Boc, Cbz, t-Bu, or trityl.

In formula (I), PG₆ is selected from the group consisting of t-Bu,trityl, cumyl, benzyl, methyl, ethyl, allyl, and2-(trimethylsilyl)ethyl.

PG₆ is preferably t-Bu, trityl, cumyl, benzyl, methyl, or allyl.

Structures represented by general formula (I), in which at least twoamino acid residues are linked to each other, include many suchstructures easily cleaved or damaged when known deprotection or resinremoval conditions are used. Only the protecting groups of interest orresins for solid-phase synthesis can be selectively and efficientlyremoved without such cleavage or damage by using the reaction conditionsof the present invention.

In some aspects, structures represented by general formula (I), in whichat least two amino acid residues are linked to each other, include thosein which one N-substituted amino acid is linked to one N-unsubstitutedamino acid and those in which two N-substituted amino acids are linkedto each other. Specific examples of such amino acid residues includestructures represented by general formula (I′), in which two amino acidresidues are linked to each other, wherein R₅ and R₈ in general formula(I) are single bonds.

Each group in formula (I′) may be the same as that in formula (I) above.

In some aspects, in formula (I′), R1 is preferably hydrogen, PG1, anatural amino acid residue, or an amino acid analog residue.

In some aspects, in formula (I′), preferably, either or both of R2 andR6 are other than hydrogen, and more preferably, either or both of R2and R6 are C1-C6 alkyl.

In some aspects, in formula (I′), R2 and R6 may be independently C1-C6alkyl, which is preferably methyl, ethyl, propyl, butyl, or pentyl.

In some aspects, in formula (I′), R2 and R4 or R2 and R4′, or R6 and R7or R6 and R7′ may each independently, together with the nitrogen atomand carbon atom to which they are attached, form a 3- to 7-memberedheterocyclic ring optionally substituted with hydroxy or C1-C4 alkoxy.R4′ is hydrogen when R2 and R4 together form the heterocyclic ring, andR4 is hydrogen when R2 and R4′ together form the heterocyclic ring. R7′is hydrogen when R6 and R7 together form the heterocyclic ring, and R7is hydrogen when R6 and R7′ together form the heterocyclic ring.

In some aspects, in formula (I′), R9 is hydroxy, —O-PG6, a natural aminoacid residue, an amino acid analog residue, —O-RES, or —NH-RES, whereRES is a resin for solid-phase synthesis.

In some aspects, in formula (I′), R4 and R4′, or R7 and R7′ may each beC1-C6 alkyl, or R4 and R4′, or R7 and R7′ may, together with the carbonatom to which they are attached, form a 3- to 7-membered alicyclic ring.

In some aspects, in formula (I′), R4′ may be hydrogen, and R4 may beselected from the group consisting of hydrogen, optionally substitutedC1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C4 alkyl, optionallysubstituted phenyl, optionally substituted phenylmethyl, optionallysubstituted phenylethyl, 2-(methylthio)ethyl, —CH2SPG2,N-PG3-indol-3-ylmethyl, 4-(PG2O)benzyl, PG2-O-methyl, 1-(PG2O)ethyl,2-(PG2O)ethyl, PG2-OCO(CH2)-, PG2-OCO(CH2)2-, PG3N-n-butyl,—CON(R14A)(R14B), —CH2-CON(R14A)(R14B), and —(CH2)2CON(R14A)(R14B).

In some aspects, in formula (I′), R7′ may be hydrogen, and R7 may beselected from the group consisting of hydrogen, optionally substitutedC1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C4 alkyl, optionallysubstituted phenyl, optionally substituted phenylmethyl, optionallysubstituted phenylethyl, 2-(methylthio)ethyl, —CH2SPG4,N-PG5-indol-3-ylmethyl, 4-(PG4O)benzyl, PG4-O-methyl, 1-(PG4O)ethyl,2-(PG4O)ethyl, PG4-OCO(CH2)-, PG4-OCO(CH2)2-, PG5N-n-butyl,—CON(R15A)(R15B), —CH2-CON(R15A)(R15B), and —(CH2)2CON(R15A)(R15B).

More preferably, in the structure represented by general formula (I′),R₁ is hydrogen, PG₁, a natural amino acid residue, or an amino acidanalog residue;

-   -   R₂ and R₆ are methyl or ethyl; and/or    -   R₂ and R₄ or R₂ and R_(4′), or R₆ and R₇ or R₆ and R_(7′) each        independently, together with the nitrogen atom and carbon atom        to which they are attached, form a 4- to 6-membered heterocyclic        ring optionally substituted with hydroxy or C₁-C₄ alkoxy. R_(4′)        is hydrogen when R₂ and R₄ together form the heterocyclic ring,        and R₄ is hydrogen when R₂ and R_(4′) together form the        heterocyclic ring. R_(7′) is hydrogen when R₆ and R₇ together        form the heterocyclic ring, and R₇ is hydrogen when R₆ and        R_(7′) together form the heterocyclic ring.    -   R₉ may be —O-PG₆, a natural amino acid residue, an amino acid        analog residue, or —O-RES, where RES is a solid-phase synthesis        CTC, Wang, or SASRIN resin.    -   R₄ and R_(4′), or R₇ and R_(7′) may be each independently methyl        or ethyl, and R₄ and R_(4′) or R₇ and R_(7′) may, together with        the nitrogen atom and carbon atom to which they are attached,        form a 5- or 6-membered alicyclic ring.        -   When R₄, and R_(7′) are hydrogen, R₄ and R₇ may be each            independently hydrogen, or alkyl selected from the group            consisting of methyl, ethyl, isopropyl, isobutyl, and            sec-butyl, or optionally substituted phenylmethyl,            optionally substituted phenylethyl, benzyloxymethyl,            1-benzyloxyethyl, 2-benzyloxyethyl,            tert-butoxycarbonylmethyl, methoxycarbonylmethyl,            tert-butoxycarbonylethyl, methoxycarbonylethyl,            tert-butoxycarbamoylbutyl, N,N-dimethylaminocarbonyl,            piperidylcarbonyl, pyrrolidylcarbonyl,            N,N-dimethylaminocarbonylmethyl, piperidylcarbonylmethyl,            pyrrolidylcarbonylmethyl, N,N-dimethylaminocarbonylethyl,            piperidylcarbonylethyl, or pyrrolidylcarbonylethyl.

In some aspects, the starting peptide compound may comprise one or moreadditional natural amino acid residues and/or amino acid analogresidues, in addition to one or more structures represented by generalformula (I).

In some aspects, the starting peptide compound comprises at least oneprotecting group removable by the method of the present invention. Sucha protecting group may be contained in a structure represented bygeneral formula (I), or may be contained in an amino acid residue otherthan the structure represented by general formula (I).

In some aspects, the starting peptide compound comprises at least oneresin for solid-phase synthesis that is removable by the method of thepresent invention. Such a resin may be contained in a structurerepresented by general formula (I), or may be contained in an amino acidresidue other than the structure represented by general formula (I).

In some aspects, the resin for solid-phase synthesis is bound to thecarboxyl group contained in the C-terminal amino acid residue of thestarting peptide compound.

In some aspects, the starting peptide compound to be subjected todeprotection and/or resin removal comprises at the C-terminus astructure in which at least two amino acid residues are linked to eachother, wherein the structure is represented by general formula (II)below.

R_(1′) is a group represented by formula (III):

-   -   wherein * represents the point of attachment.

In formula (III), R1 is hydrogen, PG1, a natural amino acid residue, oran amino acid analog residue.

In formula (III), R2 is selected from the group consisting of hydrogenand C1-C6 alkyl, or R2 and R10 or R2 and R10′, together with thenitrogen atom and carbon atom to which they are attached, form a 3- to7-membered heterocyclic ring optionally substituted with hydroxy orC1-C4 alkoxy. R10′ is hydrogen when R₂ and R₁₀ together form theheterocyclic ring, or R₁₀ is hydrogen when R₂ and R₁₀, together form theheterocyclic ring.

In some aspects, when R₂ is C₁-C₆ alkyl, the C₁-C₆ alkyl is preferablymethyl, ethyl, propyl, butyl, or pentyl.

In some aspects, when R₂ and R₁₀, or R₂ and R_(10′), together with thenitrogen atom and carbon atom to which they are attached, form a 3- to7-membered heterocyclic ring optionally substituted with hydroxy orC₁-C₄ alkoxy, the 3- to 7-membered heterocyclic ring formed ispreferably an azetidine ring, a pyrrolidine ring, or a piperidine ring.

In formula (III), except when R₂ and R₁₀ or R₂ and R_(10′) together formthe heterocyclic ring,

-   -   (a) R_(10′) is hydrogen, and R₁₀ is selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionally        substituted phenyl, optionally substituted phenylmethyl,        optionally substituted phenylethyl, 2-(methylthio)ethyl,        —CH₂SPG₈, N-PG₉-indol-3-ylmethyl, 4-(PG₈O)benzyl, PG₈-O-methyl        (i.e., —CH₂—O-PG₈), 1-(PG₈O)ethyl, 2-(PG₈O)ethyl, PG₈-OCO(CH₂)—,        PG₈-OCO(CH₂)₂—, PG₉N-n-butyl, —CON(R_(16A))(R_(16B)),        —CH₂—CON(R_(16A))(R_(16B)), and —(CH₂)₂CON(R_(16A))(R_(16B)),    -   (b) R₁₀ and R_(10′) are independently optionally substituted        C₁-C₆ alkyl, C₃-C₆ cycloalkyl, or C₃-C₆ cycloalkyl-C₁-C₄ alkyl,        or    -   (c) R₁₀ and R_(10′), together with the carbon atom to which they        are attached, form a 3- to 7-membered alicyclic ring.

The combination of R₁₀ and R_(10′) is preferably a hydrogen atom and ahydrogen atom, methyl and a hydrogen atom, ethyl and a hydrogen atom,isopropyl and a hydrogen atom, isobutyl and a hydrogen atom, cyclopropyland a hydrogen atom, cyclopropylmethyl and a hydrogen atom, optionallysubstituted phenyl and a hydrogen atom, optionally substitutedphenylmethyl and a hydrogen atom, optionally substituted phenylethyl anda hydrogen atom, 2-(methylthio)ethyl and a hydrogen atom, —CH₂SPG₈ and ahydrogen atom, N-PG₉-indol-3-ylmethyl and a hydrogen atom,4-(PG₈O)benzyl and a hydrogen atom, PG₈-O-methyl and a hydrogen atom,1-(PG₈O)ethyl and a hydrogen atom, 2-(PG₈O)ethyl and a hydrogen atom,PG₈-OCO(CH₂)— and a hydrogen atom, PG₈-OCO(CH₂)₂— and a hydrogen atom,PG₉N-n-butyl and a hydrogen atom, —CON(R_(16A))(R_(16B)) and a hydrogenatom, —CH₂—CON(R_(16A))(R_(16B)) and a hydrogen atom,—(CH₂)₂CON(R_(16A))(R_(16B)) and a hydrogen atom, methyl and methyl, ormethyl and ethyl. When R₁₀ and R_(10′), together with the carbon atom towhich they are attached, form a 3- to 7-membered alicyclic ring, thealicyclic ring is preferably a cyclopropyl ring, a cyclobutyl ring, acyclopentyl ring, or a cyclohexyl ring.

In formula (III), R₁₁ is a single bond or —C(R_(11A))(R_(11B))—; and

R_(11A) and R_(11B) are independently selected from the group consistingof hydrogen, C1-C₆ alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryl-C₁-C₄ alkyl, andoptionally substituted heteroaryl-C₁-C₄ alkyl.

R₁₁ is preferably a single bond or —C(R_(11A))(R_(11B))— where thecombination of R_(11A) and R_(11B) is a hydrogen atom and a hydrogenatom, methyl and a hydrogen atom, ethyl and a hydrogen atom, isopropyland a hydrogen atom, isobutyl and a hydrogen atom, cyclopropyl and ahydrogen atom, cyclopropylmethyl and a hydrogen atom, optionallysubstituted phenyl and a hydrogen atom, optionally substitutedphenylmethyl and a hydrogen atom, optionally substituted phenylethyl anda hydrogen atom, methyl and methyl, or methyl and ethyl.

In formula (II), R₁₂ and R_(12′) are independently selected from thegroup consisting of hydrogen, PG₁₀-O-methyl (i.e., —CH₂—O-PG₁₀),—(CH₂)_(n)COO-PG₁₀, —(CH₂)_(n)COO-RES, and —(CH₂)_(n)CONH-RES. RES is aresin for solid-phase synthesis, and n is 0, 1, or 2.

In some aspects, when used for the resin removal method of the presentinvention, either one of R12 and R12′ is preferably selected from—(CH2)nCOO-RES and —(CH2)nCONH-RES.

In formula (II), R6 is selected from the group consisting of hydrogenand C1-C6 alkyl. In some aspects, when R6 is C1-C6 alkyl, the C1-C6alkyl is preferably methyl, ethyl, propyl, butyl, or pentyl.

In formulas (II) and (III), preferably, either or both of R2 and R6 areother than hydrogen, and more preferably, either or both of R2 and R6are C1-C6 alkyl.

In formula (II), R13 is C1-C4 alkyl or —(CH2)mCON(R17A)(R17B), wherein mis 0, 1, or 2, and R17A and R17B are independently hydrogen or C1-C4alkyl, or R17A and R17B, together with the nitrogen atom to which theyare attached, form a 4- to 8-membered ring optionally comprising one ormore additional heteroatoms.

In some aspects, when R13 is C1-C4 alkyl, the C1-C4 alkyl is preferablymethyl or ethyl.

In some aspects, when R17A and R17B, together with the nitrogen atom towhich they are attached, form a 4- to 8-membered ring optionallycomprising one or more additional heteroatoms, the ring formed ispreferably an azetidine ring, a pyrrolidine ring, a piperidine ring, apiperazine ring, or a morpholine ring.

In formula (II), R16A and R16B are independently hydrogen or C1-C4alkyl, or R16A and R16B, together with the nitrogen atom to which theyare attached, form a 4- to 8-membered ring optionally comprising one ormore additional heteroatoms.

When R16A and R16B are C1-C4 alkyl, the C1-C4 alkyl is preferablymethyl, ethyl, or propyl.

When R16A and R16B, together with the nitrogen atom to which they areattached, form a 4- to 8-membered ring optionally comprising one or moreadditional heteroatoms, the 4- to 8-membered ring is preferably anazetidine ring, a pyrrolidine ring, a piperidine ring, a piperazinering, or a morpholine ring.

PG1 is selected from the group consisting of Fmoc, Boc, Alloc, Cbz,Teoc, and trifluoroacetyl.

PG₁ is preferably Fmoc, Boc, or Cbz.

In formula (II), PG₈ is selected from the group consisting of hydrogen,t-Bu, trityl, methoxytrityl, cumyl, benzyl, THP, 1-ethoxyethyl, methyl,ethyl, allyl, optionally substituted aryl, optionally substitutedaryl-C₁-C₄ alkyl, optionally substituted heteroaryl-C₁-C₄ alkyl, and2-(trimethylsilyl)ethyl.

PG₈ is preferably methyl, allyl, t-Bu, trityl, methoxytrityl, cumyl,THP, optionally substituted aryl, optionally substituted aryl-C₁-C₄alkyl, or optionally substituted heteroaryl-C₁-C₄ alkyl.

In formula (II), PG9 is selected from the group consisting of hydrogen,Fmoc, Boc, Alloc, Cbz, Teoc, methoxycarbonyl, t-Bu, trityl, cumyl, andbenzyl. PG9 is preferably Fmoc, Boc, Cbz, t-Bu, or trityl.

In formula (II), PG10 is selected from the group consisting of t-Bu,trityl, cumyl, benzyl, methyl, ethyl, allyl, optionally substitutedaryl, optionally substituted aryl-C1-C4 alkyl, optionally substitutedheteroaryl-C1-C4 alkyl, and 2-(trimethylsilyl)ethyl. PG10 is preferablyt-Bu, trityl, cumyl, benzyl, methyl, or allyl.

In some aspects, PG1 to PG10 that can be comprised in general formulas(I) to (III) may be each independently a protecting group that can beremoved by the deprotection method of the present invention. Suchprotecting groups include t-Bu, triphenylmethyl,2-(trimethylsilyl)-ethyl, Boc, Teoc, Cbz, methoxycarbonyl,tetrahydropyranyl, and 1-ethoxyethyl.

In some aspects, PG1 to PG10 may be each independently a protectinggroup that cannot be removed by the deprotection method of the presentinvention. Such protecting groups include Fmoc and Alloc.

In some aspects, PG1 to PG10 may be each independently a group that doesnot function as a protecting group, for example, a group that cannot beremoved or can be structurally transformed. Examples of such protectinggroups include those described in Greene's “Protective Groups in OrganicSynthesis” (5th ed., John Wiley & Sons 2014).

Structures represented by general formula (II), in which at least twoamino acid residues are linked to each other, include many suchstructures easily subjected to cleavage or rearrangement when knowndeprotection or resin removal conditions are used. Such cleavage andrearrangement may be called damage. Only the protecting groups ofinterest or resins for solid-phase synthesis can be selectively andefficiently removed without such damage using the reaction conditions ofthe present invention.

In some aspects, structures represented by general formula (II), inwhich two amino acid residues are linked to each other, include those inwhich one N-substituted amino acid is linked to one N-unsubstitutedamino acid and those in which two N-substituted amino acids are linkedto each other. Specific examples of such structures include structuresin which R1′ of general formula (II) is represented by general formula(III) and in which R₁₁ of formula (III) is —C(R_(11A))(R_(11B))—(formula IV) or R₁₁ of formula (III) is a single bond (formula V).

Each group in formulas (IV) and (V) may be the same as that in formulas(II) and (III) above.

In some aspects, in formulas (IV) and (V), R1 is hydrogen, PG1, anatural amino acid residue, or an amino acid analog residue.

In some aspects, in formulas (IV) and (V), R1 is hydrogen, PG1, anatural amino acid residue, or an amino acid analog residue, and R2 andR10 are preferably methyl, ethyl, propyl, butyl, or pentyl.

In some aspects, in formulas (IV) and (V), R1 is hydrogen, PG1, anatural amino acid residue, or an amino acid analog residue, and R2 andR10 or R2 and R10′ preferably each independently, together with thenitrogen atom and carbon atom to which they are attached, form a 3- to7-membered heterocyclic ring optionally substituted with hydroxy orC1-C4 alkoxy. R10′ is hydrogen when R2 and R10 together form theheterocyclic ring, and R10 is hydrogen when R2 and R10′ together formthe 3- to 7-membered heterocyclic ring.

In some aspects, in formulas (IV) and (V), R10′ may be hydrogen, and R10may be selected from the group consisting of hydrogen, optionallysubstituted C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl-C1-C4 alkyl,optionally substituted phenyl, optionally substituted phenylmethyl,optionally substituted phenylethyl, 2-(methylthio)ethyl, —CH2SPG8,N-PG9-indol-3-ylmethyl, 4-(PG8O)benzyl, PG8-O-methyl, 1-(PG8O)ethyl,2-(PG8O)ethyl, PG8-OCO(CH2)-, PG8-OCO(CH2)2-, PG9N-n-butyl,—CON(R16A)(R16B), —CH2-CON(R16A)(R16B), and —(CH2)2CON(R16A)(R16B).

In some aspects, in formulas (IV) and (V), preferably, R6 may be C1-C6alkyl, which is preferably methyl, ethyl, propyl, butyl, or pentyl.

In some aspects, in formulas (IV) and (V), R13 may be C1-C4 alkyl, whichis preferably methyl, ethyl, propyl, or butyl, and more preferablymethyl or ethyl.

In some aspects, in formulas (IV) and (V), R₁₃ may be—(CH₂)_(m)CON(R_(17A))(R_(17B)), and m is 0, 1, or 2. In this case, R17Aand/or R17B may be independently C1-C4 alkyl, and the C1-C4 alkyl ispreferably methyl or ethyl. R17A and R17B may, together with thenitrogen atom to which they are attached, form a 4- to 8-membered ringoptionally comprising one or more additional heteroatoms, and the 4- to8-membered ring is preferably an azetidine ring, a pyrrolidine ring, apiperidine ring, a piperazine ring, or a morpholine ring.

In some aspects, in formulas (IV) and (V), R12 and R12′ areindependently selected from the group consisting of hydrogen,PG10-O-methyl, —(CH2)nCOO-PG10, —(CH2)nCOO-RES, and —(CH2)nCONH-RES.When R12 and R12′ is PG10-O-methyl, —(CH2)nCOO-PG10, —(CH2)nCOO-RES, or—(CH2)nCONH-RES, n is 0, 1, or 2, and PG10 is preferably t-Bu, trityl,cumyl, benzyl, methyl, or allyl. RES is a resin for solid-phasesynthesis, and preferably CTC resin or Wang resin.

In some aspects, when used for the resin removal method of the presentinvention, either one of R12 and R12′ is preferably selected from—(CH2)nCOO-RES and —(CH2)nCONH-RES.

In some aspects, in formulas (IV) and (V), more preferably, R1 ishydrogen, PG₁, a natural amino acid residue, or an amino acid analogresidue;

-   -   R₂ is methyl and ethyl; and/or    -   R₂ and R₁₀, together with the nitrogen atom and carbon atom to        which they are attached, form a 4- to 6-membered heterocyclic        ring optionally substituted with hydroxy or C₁-C₄ alkoxy.    -   R₁₀ and R_(10′) may be each independently selected from methyl        or ethyl, or R₁₀ and R_(10′) may together form a 5- or        6-membered alicyclic ring. Alternatively, R_(10′) may be        hydrogen, and R₁₀ may be hydrogen, or alkyl selected from        methyl, ethyl, isopropyl, isobutyl, and sec-butyl, or optionally        substituted phenylmethyl, optionally substituted phenylethyl,        benzyloxymethyl, 1-benzyloxyethyl, 2-benzyloxyethyl,        tert-butoxycarbonylmethyl, methoxycarbonylmethyl,        tert-butoxycarbonylethyl, methoxycarbonylethyl,        tert-butoxycarbamoylbutyl, N,N-dimethylaminocarbonyl,        piperidylcarbonyl, pyrrolidylcarbonyl,        N,N-dimethylaminocarbonylmethyl, piperidylcarbonylmethyl,        pyrrolidylcarbonylmethyl, N,N-dimethylaminocarbonylethyl,        piperidylcarbonylethyl, or pyrrolidylcarbonylethyl.    -   R₆ may be methyl or ethyl, and    -   R₁₃ may be methyl or —(CH₂)_(m)CON(R_(17A))(R_(17B)). When R₁₃        is —(CH₂)_(m)CON(R_(17A))(R_(17B)), R_(17A) and R_(17B),        together with the nitrogen atom to which they are attached, form        a piperidine ring.    -   R₁₂ and R_(12′) are independently selected from the group        consisting of hydrogen, PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀,        —(CH₂)_(n)COO-RES, and —(CH₂)_(n)CONH-RES.

In some aspects, the starting peptide compound may comprise one or moreadditional natural amino acid residues and/or amino acid analogresidues, in addition to a structure represented by general formula(II). The starting peptide compound may comprise one or more structuresrepresented by general formula (I), may comprise a structure representedby general formula (II) at the C-terminus, and optionally may furthercomprise one or more additional natural amino acid residues and/or aminoacid analog residues. The peptide compound may be formed by secondaryamides, tertiary amides, or a mixture of secondary and tertiary amides.

In some aspects, the starting peptide compound comprises at least oneprotecting group removable by the method of the present invention. Sucha protecting group may be contained in a structure represented bygeneral formula (II), or may be contained in an amino acid residue otherthan the structure represented by general formula (II).

In some aspects, the starting peptide compound comprises at least oneresin for solid-phase synthesis that is removable by the method of thepresent invention. When the starting peptide compound comprises astructure represented by general formula (II), such a resin is includedin the structure represented by general formula (II).

In some aspects, the present invention relates to a method of producingan amide compound in which a protecting group removable by a silylatingagent is removed, the method comprising the step of contacting astarting amide compound with the silylating agent in a solvent andthereby removing the protecting group from the starting amide compound.

In some aspects, the present invention relates to a method of producingan amide compound in which a resin for solid-phase synthesis is removed,the method comprising the step of contacting a starting amide compoundwith a silylating agent in a solvent and thereby removing the startingamide compound from the resin for solid-phase synthesis

In some aspects, the starting amide compound to be subjected todeprotection/resin removal is represented by general formula (II).

In formula (II), R_(1′) is a hydrogen atom or PG₇, and the other groupsare the same as defined above. R₆ is preferably C₁-C₆ alkyl. PG₇ isselected from the group consisting of Fmoc, Boc, Alloc, Cbz, Teoc, andtrifluoroacetyl and is preferably Fmoc, Boc, or Cbz.

Starting amide compounds represented by general formula (II), inparticular, N-substituted compounds are susceptible to damage such asrearrangement when known deprotection or resin removal conditions areused. Only the protecting groups of interest or resin for solid-phasesynthesis can be selectively and efficiently removed without such damageusing the reaction conditions of the present invention.

Herein, the “silylating agent” is not particularly limited as long as itcan be used for the deprotection or resin removal reaction of thepresent invention, and the term refers to an agent that can function asa deprotecting agent or reagent and/or a resin-removing agent orreagent. Herein, “silylating agents” may be called deprotecting agentsor reagents and/or resin-removing agents or reagents. The silylatingagent can be prepared, for example, by mixing the silyl compound with anelectrophilic species scavenger or mixing an acid with an electrophilicspecies scavenger having a silyl group in a solvent. The mixing of thesilyl compound with an electrophilic species scavenger or the mixing ofan acid with an electrophilic species scavenger having a silyl group maybe performed previously, or in a solvent, or in the presence or absenceof the starting peptide compound.

Examples of the “silyl compound” as used herein include silyl compoundshaving a leaving group (X) represented by formula 1:

wherein R_(AX), R_(AY), and R_(AZ) are independently C₁-C₄ alkyl orphenyl, and X is selected from the group consisting of —OTf, —OClO₃, Cl,Br, and I.Preferably, R_(AX), R_(AY), and R_(AZ) may be independently selectedfrom methyl, ethyl, i-propyl, t-butyl, and phenyl.

More specific examples of such silyl compounds include TMSOTf, TESOTf,TBSOTf, TIPSOTf, TBDPSOTf, TTMSOTf, TMSCl, TMSBr, TMSOClO₃, and TMSI.

The “acid” that can be used herein for preparing a silylating agent isnot particularly limited as long as it can generate the silyl compound,and examples thereof include acids represented by HX (wherein X is asdefined for X of formula (1)).

As used herein, the term “electrophilic species scavenger” refers to acompound that can form an adduct or salt with a proton or is reactivewith a cationic species and that is less susceptible to silylation. Suchelectrophilic species scavengers include imidates (formula 2), amides(formula 3), ketene acetals (formula 4), ketene alkoxy hemiaminals(formula 4), enol ethers (formula 4), enol esters (formula 4), imines(formula 5), amines (formula 6), diamines (formula 7),dialkylcarbodiimides (formula 8), ureas (formula 9), or urethanes(formula 10). They may have a substituted silyl group, specific examplesof the substituted silyl group include TMS (trimethylsilyl), TES(triethylsilyl), TBS (tributylsilyl), TIPS (triisopropylsilyl), andTBDPS (t-butyl-dimethylsilyl).

The imidates are represented by formula 2:

wherein

-   -   R_(B) is a substituted silyl group and R_(C) is a substituted        silyl group, or    -   R_(B) and R_(C), together with the nitrogen atom and carbon atom        to which they are attached, form a 5- to 7-membered ring; and    -   R_(D) is C₁-C₄ alkyl optionally substituted with one or more        fluorine atoms or is optionally substituted methylene, wherein        when R_(D) is optionally substituted methylene, formula 2 is        dimerized to form a compound represented by the formula below:

Preferred imidates include N-silyl imidates represented by formula 2-1and bisoxazolines represented by formula 2-2 below:

wherein

-   -   R_(BX), R_(BY), and R_(BZ) are independently C₁-C₄ alkyl or        phenyl, R_(C) and R_(D) are as defined above; and    -   R_(E) and R_(F) are independently C₁-C₄ alkyl.    -   Preferably, R_(BX), R_(BY), and R_(BZ) may be independently        selected from methyl, ethyl, i-propyl, t-butyl, and phenyl.

Preferred N-silyl imidates include N,O-bis(trimethylsilyl)acetamidesrepresented by formula 2-1-1. Preferred bisoxazolines include4-substituted bisoxazolines represented by formula 2-2-1.

More preferred N-silyl imidates include N,O-bis(trimethylsilyl)acetamiderepresented by formula 2-1-1-1 andN,O-bis(trimethylsilyl)trifluoroacetamide represented by formula2-1-1-2. More preferred bisoxazolines includeN,O-bis(trimethylsilyl)trifluoroacetamide2,2′-isopropylidenebis[(4S)-4-tert-butyl-2-oxazoline] represented byformula 2-2-1-1.

The amides are represented by formula 3:

wherein

-   -   R_(G) is a silyl group substituted with one or more C₁-C₄ alkyl;    -   R_(H) is hydrogen or C₁-C₄ alkyl; and    -   R_(I) is hydrogen, or C₁-C₄ alkyl optionally substituted with        one or more fluorine atoms.

Preferred amides include N-silylamides represented by formula 3-1:

wherein

-   -   RGX, RGY, and RGZ are independently C1-C4 alkyl or phenyl; and    -   R_(H) and R_(I) are as defined above.

Preferably, R_(GX), R_(GY), and R_(GZ) may be independently selectedfrom methyl, ethyl, i-propyl, t-butyl, and phenyl.

Preferred N-silylamides include N-trimethylsilylacetamides representedby formula 3-1-1:

wherein RH and RI are as defined above.

More preferably, N-trimethylsilylacetamides includeN-methyl-N-trimethylsilylacetamide represented by formula 3-1-1-1 andN-methyl-N-trimethylsilyltrifluoroacetamide represented by formula3-1-1-2.

The ketene acetals, ketene alkoxy hemiaminals, enol ethers, and enolesters are each represented by formula 4:

wherein

-   -   (a-1) RJ is a substituted silyl group, RK is C1-C4 alkoxy, and        RM and RL are independently hydrogen or C₁-C₄ alkyl;    -   (a-2) R_(J) is a substituted silyl group, R_(M) is hydrogen or        C₁-C₄ alkyl, and R_(K) and R_(L), together with the carbon atoms        to which they are attached, form a 5- to 8-membered ring        comprising an oxygen atom;    -   (b-1) R_(J) is a substituted silyl group, R_(K) is C₁-C₄ alkyl,        and R_(M) and R_(L) are independently hydrogen or C₁-C₄ alkyl;    -   (b-2) R_(J) is a substituted silyl group, R_(M) is hydrogen or        C₁-C₄ alkyl, and R_(K) and R_(L), together with the carbon atoms        to which they are attached, form a 5- to 8-membered ring;    -   (c-1) R_(J) and R_(M), together with the carbon atoms to which        they are attached, form a 5- to 7-membered ring comprising an        oxygen atom, R_(K) is hydrogen or C₁-C₄ alkyl, and R_(L) is        C₁-C₄ alkyl;    -   (c-2) R_(J) and R_(M), together with the carbon atoms to which        they are attached, form a 5- to 7-membered ring comprising an        oxygen atom, and R_(K) and R_(L), together with the carbon atoms        to which they are attached, form a 5- to 8-membered ring;    -   (d-1) R_(J) is C₁-C₄ alkyl and R_(M), R_(K), and R_(L) are        independently hydrogen or C₁-C₄ alkyl;    -   (d-2) R_(J) is C₁-C₄ alkyl, R_(M) is hydrogen or C₁-C₄ alkyl,        and R_(K) and R_(L), together with the carbon atoms to which        they are attached, form a 5- to 8-membered ring;    -   (e-1) R_(J) is C₁-C₃ alkylcarbonyl and R_(M), R_(K), and R_(L)        are independently hydrogen or C1-C4 alkyl;    -   (e-2) R_(J) is C₁-C₃ alkylcarbonyl, R_(M) is hydrogen or C1-C4        alkyl, and R_(K) and R_(L), together with the carbon atoms to        which they are attached, form a 5- to 8-membered ring;    -   (f-1) RJ is a substituted silyl group or C1-C4 alkyl, RK is        optionally substituted di-C1-C4 alkylamino, and RM and RL are        independently hydrogen or C1-C4 alkyl; or    -   (f-2) RJ is a substituted silyl group or C1-C4 alkyl, RM is        hydrogen or C1-C4 alkyl, and RK and RL, together with the carbon        atoms to which they are attached, form a 5- to 8-membered ring        comprising a nitrogen atom, and the 5- to 8-membered ring is        optionally substituted with C₁-C₄ alkyl.

Here, preferred examples of the C₁-C₄ alkyl include methyl, ethyl,n-propyl, isopropyl, n-butyl, i-butyl, and t-butyl. Preferred examplesof the C₁-C₃ alkylcarbonyl include acetyl and propionyl.

Preferred ketene acetals include silylketene acetals represented byformula 4-1:

wherein

-   -   R_(JX1), R_(JY1), and R_(JZ1) are independently C₁-C₄ alkyl or        phenyl;    -   R_(KX1) is C₁-C₄ alkyl; and    -   R_(L) and R_(M) are as defined above.    -   Here, preferred examples of the C₁-C₄ alkyl include methyl,        ethyl, n-propyl, isopropyl, n-butyl, i-butyl, and t-butyl.    -   Preferably, R_(JX1), R_(JY1), and R_(JZ1) may be independently        selected from methyl, ethyl, i-propyl, t-butyl, and phenyl.

Preferred ketene silyl acetals include ketene trimethylsilyl acetalsrepresented by formula 4-1-1:

wherein R_(KX1), R_(L), and R_(M) are as defined above.

Preferred R_(KX1), R_(L), and R_(M) include methyl, ethyl, n-propyl,isopropyl, n-butyl, i-butyl, and t-butyl.

Preferred ketene trimethylsilyl acetals include dimethylketene methyltrimethylsilyl acetal represented by formula 4-1-1-1.

Preferred enol ethers include silylenol ethers represented by formula4-2:

wherein

-   -   R_(JX2), R_(JY2), and R_(JZ2) are independently C₁-C₄ alkyl or        phenyl;    -   R_(KX2) is hydrogen or C₁-C₄ alkyl; and    -   R_(L) and R_(M) are as defined above.    -   Preferred C₁-C₄ alkyl of R_(KX1), R_(L), and R_(M) include        methyl, ethyl, n-propyl, isopropyl, n-butyl, i-butyl, and        t-butyl.    -   Preferably, R_(JX2), R_(JY2), and R_(JZ2) are independently        selected from the group consisting of methyl, ethyl, i-propyl,        t-butyl, and phenyl.

Preferred silyl enol ethers include trimethylsilyl enol ethersrepresented by formula 4-2-1:

wherein

-   -   R_(KX2), R_(L), and R_(M) are as defined above.

Preferred trimethylsilyl enol ethers includeisopropenyloxytrimethylsilane represented by formula 4-2-1-1.

In another embodiment, preferred enol ethers include cyclic enol ethersrepresented by formula 4-3:

wherein

-   -   R_(KX3) and RU are independently hydrogen or C₁-C₄ alkyl.

Preferred cyclic enol ethers include dihydropyran represented by formula4-3-1.

In another embodiment, preferred enol ethers include linear enol ethers,and preferred linear enol ethers include ethyl vinyl ether representedby formula 4-4.

The imines are represented by formula 5:

wherein

-   -   R_(N), R_(N′), and R_(O) are independently hydrogen or C₁-C₄        alkyl.    -   Preferred C₁-C₄ alkyl of R_(N), R_(N′), and R_(O) include        methyl, ethyl, n-propyl, isopropyl, n-butyl, i-butyl, and        t-butyl.

Preferred imines include ketimines represented by formula 5-1:

-   -   wherein R_(N) is as defined above.    -   Preferred R_(N) and R_(N′) include methyl, ethyl, n-propyl,        isopropyl, n-butyl, i-butyl, and t-butyl.

Preferred ketimines include 2,2,4,4-tetramethylpentanone iminerepresented by formula 5-1-1.

The amines are represented by formula 6:

-   -   wherein        -   R_(P) is a substituted silyl group; and        -   R_(Q) is a substituted silyl group or C₁-C₄ alkyl and R_(R)            is hydrogen, a substituted silyl group, or C₁-C₄ alkyl, or        -   R_(Q) and R_(R), together with the nitrogen atom to which            they are attached, form a 5- to        -   8-membered heterocyclic ring comprising an oxygen atom.

Preferred amines include disilylamines represented by formula 6-1:

-   -   wherein        -   R_(PX1), R_(PY1), R_(PZ1), R_(QX1), R_(QY1), and R_(QZ1) are            independently C₁-C₄ alkyl or phenyl.        -   Preferably, R_(PX1), R_(PY1), R_(PZ1), R_(QX1), R_(QY1), and            R_(QZ1) are independently selected from the group consisting            of methyl, ethyl, i-propyl, t-butyl, and phenyl.

Preferred disilylamines include 1,1,1,3,3,3-hexamethyldisilazane (HMDS)represented by formula 6-1-1.

In another embodiment, preferred amines include N-dialkyl-N-silylaminesrepresented by formula 6-2:

-   -   wherein        -   R_(PX2), R_(PY2), and R_(PZ2) are independently C₁-C₄ alkyl            or phenyl; and        -   R_(Q) and R_(R) are as defined above.        -   Preferred C₁-C₄ alkyl of R_(PX2), R_(PY2), and R_(PZ2)            include methyl, ethyl, n-propyl, isopropyl, n-butyl,            i-butyl, and t-butyl.        -   Preferably, R_(PX2), R_(PY2), and R_(PZ2) are independently            selected from the group consisting of methyl, ethyl,            i-propyl, t-butyl, and phenyl.

Preferred N-dialkyl-N-silylamines includeN-dialkyl-N-trimethylsilylamines represented by formula 6-2-1:

wherein R_(Q) and R_(R) are as defined above.

Preferred N-dialkyl-N-trimethylsilylamines includeN-trimethylsilylmorpholine represented by formula 6-2-1-1 andN-trimethylsilyldiethylamine represented by formula 6-2-1-2.

In another embodiment, preferred amines include N-alkyl-N-disilylaminesrepresented by formula 6-3:

-   -   wherein        -   R_(QX3), R_(QY3), R_(QZ3), R_(RX1), R_(RY1), and R_(RZ1) are            independently C₁-C₄ alkyl or phenyl; and        -   R_(R) is C₁-C₄ alkyl.        -   Specifically, for example, R_(QX3), R_(QY3), R_(QZ3),            R_(RX1), R_(RY1), and R_(RZ1) may be independently selected            from methyl, ethyl, i-propyl, t-butyl, and phenyl.        -   Preferred C₁-C₄ alkyl of R_(R) include methyl, ethyl,            n-propyl, isopropyl, n-butyl, i-butyl, and t-butyl.

Preferred N-alkyl-N-disilylamines includeN-alkyl-N-bistrimethylsilylamines represented by formula 6-3-1:

-   -   wherein        -   R_(R) is C₁-C₄ alkyl.        -   Preferred C₁-C₄ alkyl of R_(R) include methyl, ethyl,            n-propyl, isopropyl, n-butyl, i-butyl, and t-butyl.

In another embodiment, preferred amines include N-alkyl-N-silylaminesrepresented by formula 6-4:

-   -   wherein        -   R_(PX2), R_(PY2), and R_(PZ2) are independently C₁-C₄ alkyl            or phenyl; and        -   R_(Q) is C₁-C₄ alkyl.        -   Preferably, R_(PX2), R_(PY2), and R_(PZ2) are independently            selected from the group consisting of methyl, ethyl,            i-propyl, t-butyl, and phenyl.        -   Preferred C₁-C₄ alkyl of R_(Q) include methyl, ethyl,            n-propyl, isopropyl, n-butyl, i-butyl, and t-butyl.

Preferred N-alkyl-N-silylamines include N-alkyl-N-trimethylsilylaminesrepresented by formula 6-4-1:

-   -   wherein R_(Q) is C₁-C₄ alkyl.    -   Preferred C1-C4 alkyl of R_(Q) include methyl, ethyl, n-propyl,        isopropyl, n-butyl, i-butyl, and t-butyl.

Preferred N-alkyl-N-trimethylsilylamines includeN-tert-butyltrimethylsilylamine represented by formula 6-4-1-1.

The diamines are represented by formula 7:

-   -   wherein        -   X is a single bond or a carbon atom,        -   wherein when X is a single bond, R_(S) is absent, R_(UA) and            R_(R), together with the carbon atom and nitrogen atom to            which they are attached, form an optionally substituted            6-membered aromatic heterocyclic ring, and R_(UB) and R_(T),            together with the carbon atom and nitrogen atom to which            they are attached, form an optionally substituted 6-membered            aromatic heterocyclic ring, and        -   when X is a carbon atom, R_(UA) and R_(UB) are independently            C₁-C₄ alkyl and R_(R), R_(S), and R_(T), together with the            carbon atoms to which they are attached, form the structure            below:

When X in formula 7 is a carbon atom, preferred diamines includetetraalkylnaphthalenediamines represented by formula 7-1:

-   -   wherein R_(UA) and R_(UB) are independently C₁-C₄ alkyl.

Preferred tetraalkylnaphthalenediamines includeN,N,N′,N′-tetramethyl-1,8-naphthalenediamine represented by formula7-1-1.

In another embodiment, when X in formula 7 is a single bond, preferreddiamines include 2,2′-bipyridine represented by formula 7-2-1.

The dialkylcarbodiimides are represented by formula 8:

-   -   wherein R_(V) is C₁-C₄ alkyl or C₃-C₆ cycloalkyl.        -   Preferred C₁-C₄ alkyl of R_(V) include methyl, ethyl,            n-propyl, isopropyl, n-butyl, i-butyl, t-butyl, and            cyclohexyl.

Preferred dialkylcarbodiimides include diisopropylcarbodiimiderepresented by formula 8-1.

The ureas are represented by formula 9:

-   -   wherein R_(W) and R_(X) are independently C1-C4 alkyl or a        substituted silyl group.

Preferred ureas include N,N′-bis(trimethylsilyl)urea represented byformula 9-1.

The urethanes are represented by formula 10:

-   -   wherein R_(Y) and R_(Z) are independently C₁-C₄ alkyl or a        substituted silyl group.

Preferred urethanes include N,O-bis(trimethylsilyl)urea represented byformula 10-1.

In some aspects, silylating agents can be prepared by mixing a peptidecompound, a solvent, an electrophilic species scavenger, and a silylcompound or acid in any order. It is preferred to mix the peptidecompound with the solvent, then with the electrophilic speciesscavenger, and subsequently with the silyl compound or acid.

In some aspects, the solvent used may be an aprotic solvent, examples ofwhich include esters, ethers, alkylnitriles, halogenated hydrocarbons,and hydrocarbons. Among them, ethyl acetate, isopropyl acetate,2-methyltetrahydrofuran, tetrahydrofuran, diethyl ether, methyltert-butyl ether, dichloromethane, 1,2-dichloroethane, toluene, oracetonitrile is preferred, ethyl acetate, isopropyl acetate,2-methyltetrahydrofuran, tetrahydrofuran, or 1,2-dichloroethane is morepreferred, and ethyl acetate or 2-methyltetrahydrofuran is particularlypreferred.

In some aspects, a stoichiometric amount or more of a silyl compound,and an electrophilic species scavenger can be used in order to produce asilylating agent in the present invention to remove a protecting groupand/or remove a resin from a peptide compound. In this case, 1 to 5equivalents, preferably 2 to 4 equivalents, of a silyl compound and 1 to10 equivalents, preferably 1 to 8 equivalents, of an electrophilicspecies scavenger can be used for one equivalent of the protecting groupto be removed or one equivalent of the resin to be removed which iscontained in a peptide, for example.

In some aspects, a silylating agent can be prepared by combining anelectrophilic species scavenger with a catalytic amount of, for example,0.1 to 0.5 equivalent, preferably 0.3 to 0.4 equivalent, of a silylcompound, per one equivalent of the protecting group to be removed orone equivalent of the resin to be removed. In this case, TMSOTf, TESOTf,TBSOTf, TIPSOTf, TBDPSOTf, TTMSOTf, TMSCl, TMSBr, or TMSOClO₃ ispreferably used as such a silylating agent, andN,O-bis(trimethylsilyl)acetamide,N,O-bis(trimethylsilyl)trifluoroacetamide,N-methyl-N-trimethylsilylacetamide,N-methyl-N-trimethylsilyltrifluoroacetamide, dimethylketene methyltrimethylsilyl acetal, or isopropenyloxytrimethylsilane is preferablyused as such an electrophilic species scavenger. In this case, 1 to 10equivalents, preferably 1 to 8 equivalents, of the electrophilic speciesscavenger can be used per one equivalent of the protecting group to beremoved or one equivalent of the resin to be removed.

In some aspects, a silylating agent can be prepared by combining anelectrophilic species scavenger with a catalytic amount of, for example,0.1 to 0.5 equivalent, preferably 0.3 to 0.4 equivalent, of an acid perone equivalent of the protecting group to be removed or one equivalentof the resin to be removed. In this case, TfOH, HOClO₃, HCl, HBr, or HIis preferably used, and TfOH is more preferably used as such an acid.N-silyl imidates (formula 2-1), N-silylamides (formula 3-1), ketenesilyl acetals (formula 4-1), and silyl enol ethers (formula 4-2) arepreferably used, and N,O-bis(trimethylsilyl)acetamide (formula 2-1-1-1),N,O-bis(trimethylsilyl)trifluoroacetamide (formula 2-1-1-2),N-methyl-N-trimethylsilylacetamide (formula 3-1-1-1),N-methyl-N-trimethylsilyltrifluoroacetamide (formula 3-1-1-2),dimethylketene methyl trimethylsilyl acetal (formula 4-1-1-1), andisopropenyloxytrimethylsilane (formula 4-2-1-1) are more preferably usedas such electrophilic species scavengers. Among them,N,O-bis(trimethylsilyl)trifluoroacetamide andN-methyl-N-trimethylsilyltrifluoroacetamide are particularly preferred.In this case, 1 to 10 equivalents, preferably 1 to 8 equivalents, of theelectrophilic species scavenger can be used per one equivalent of theprotecting group to be removed or one equivalent of the resin to beremoved.

In some aspects, the “protecting group removable by a silylating agent”is not particularly limited, as long as the protecting group can beremoved by the silylating agent. Specific examples of such protectinggroups include protecting groups for carboxyl groups, such as t-Bu,benzyl, triphenylmethyl, cumyl, and 2-(trimethylsilyl)-ethyl; protectinggroups for amino groups, such as Boc, Teoc, Cbz, methoxycarbonyl, andcumyl; protecting groups for hydroxy groups, such as tetrahydropyranyland 1-ethoxyethyl; and protecting groups for SH groups, such astriphenylmethyl and methoxytrityl.

Chemical synthesis methods for start peptide compounds herein include,for example, liquid phase synthesis methods, solid phase synthesismethods using Fmoc synthesis, Boc synthesis, or such, and combinationsthereof. In Fmoc synthesis, an amino acid in which the main chain aminogroup is protected with an Fmoc group, the side-chain functional groupsare protected when necessary with protecting groups that are not cleavedby a base such as piperidine, and the main chain carboxylic acid groupis not protected, is used as a basic unit. The basic unit is notparticularly limited and may be any other combination as long as it hasan Fmoc-protected amino group and a carboxylic acid group. For example,a dipeptide may be used as a basic unit. The basic unit to be positionedat the N terminus may be one that is not an Fmoc amino acid. Forexample, it may be a Boc amino acid, or a carboxylic acid analog thatdoes not have an amino group. The main chain carboxylic acid group isimmobilized onto a solid phase by a chemical reaction with a functionalgroup on a solid-phase carrier. Next, the Fmoc group is deprotected by abase such as piperidine or DBU, and the newly generated amino group anda subsequently added basic unit, i.e. a protected amino acid carrying acarboxylic acid, are subjected to a condensation reaction to generate anamide bond. In the condensation reaction, various combinations such asDIC and HOBt, DIC and HOAt, and HATU and DIPEA are possible. Repeatingthe Fmoc group deprotection and the subsequent amide bond-formingreaction enables generation of the desired peptide sequence. After thedesired sequence is obtained, removal of the resin from the solid phaseis performed, and the protecting groups introduced as necessary to theside-chain functional groups are deprotected. The deprotection and resinremoval methods of the present invention can be provided for removalfrom solid-phase resins and removal of protecting groups from side chainfunctional groups. Steps such as cyclization may also be conductedduring the solid-phase resin removal step or the deprotection step orfinally. For example, side chain carboxylic acids may be condensed withN-terminal amino groups of main chains, and side chain amino groups maybe condensed with C-terminal carboxylic acids of main chains. In thiscase, reaction orthogonality is needed between the C-terminal carboxylicacid and the side chain carboxylic acid to be cyclized, or between theN-terminal amino or hydroxy group of the main chain and the side chainamino group to be cyclized, and the protecting group is selected takingorthogonality of the protecting group into consideration. Reactionproducts thus obtained can be purified by reverse phase columns,molecular sieve columns, or the like. The details are described inSolid-Phase Synthesis Handbook published by Merck K.K. on May 1, 2002,for example.

Herein, the “resin for solid-phase synthesis” is not particularlylimited, as long as it can be used for the synthesis of peptidecompounds by the solid-phase method and can be removed by the silylatingagent of the present invention. Specific examples of such resins forsolid-phase synthesis include those removable under acidic conditions,such as CTC resins, Wang resins, SASRIN resins, trityl chloride resins(Trt resins), 4-methyltrityl chloride resins (Mtt resins), and4-methoxytrityl chloride resins (Mmt). The resin can be appropriatelyselected according to the functional group on the amino acid to be used.For example, when using a carboxylic acid (main-chain carboxylic acid orside-chain carboxylic acid represented by Asp or Glu) or a hydroxy groupon an aromatic ring (phenol group represented by Tyr) as the functionalgroup on the amino acid, use of trityl chloride resin (Trt resin) or2-chlorotritylchloride resin (CTC resin) as the resin is preferred. Whenusing an aliphatic hydroxy group (aliphatic alcohol group represented bySer or Thr) as the functional group on the amino acid, use oftritylchloride resin (Trt resin), 2-chlorotritylchloride resin (CTCresin), or 4-methyltritylchloride resin (Mtt resin) as the resin ispreferred.

Furthermore, the types of polymers constituting the resins are also notparticularly limited. For resins composed of polystyrenes, either 100 to200 mesh or 200 to 400 mesh may be used. The cross-link percentage isalso not particularly limited, but those cross-linked with 1%divinylbenzene (DVB) are preferred. Types of the polymer forming theresin include Tentagel or Chemmatrix.

In some aspects, the deprotection reaction and/or resin removal reactionof the present invention can be performed at a reaction temperature of−50 to 100° C., preferably −20 to 50° C., and more preferably 0 to 30°C.

In some aspects, the deprotection reaction and/or resin removal reactionof the present invention can be performed for a reaction time of 10minutes to one week, preferably 10 minutes to 6 hours, and morepreferably 1 to 3 hours.

In some aspects, in the deprotection reaction and/or resin removalreaction of the present invention, a target product can be obtained byadding an alcohol or water to a reaction solution to quench thereaction, and causing the target product to precipitate or washing anorganic layer with water or saline and then concentrating the organiclayer under reduced pressure. The alcohol is not particularly limited,but is preferably a water-soluble and low-boiling alcohol, andparticularly preferably methanol. The water used for quenching thereaction is not particularly limited, but is preferably alkaline water,and particularly preferably aqueous sodium bicarbonate or dipotassiumhydrogenphosphate. When the organic layer is washed, the saline is notparticularly limited in terms of concentration, but is preferably brineor 5% saline.

In some aspects, when a starting peptide compound comprises both aprotecting group that can be removed by the method of the presentinvention and a resin for solid-phase synthesis that can be removed bythe method of the present invention, deprotection and resin removalreactions can be conducted at the same time. Specifically, the presentinvention also relates to a method of producing a peptide compound inwhich a protecting group removable by a silylating agent is removed froma starting peptide compound comprising natural amino acid residuesand/or amino acid analog residues and in which a resin for solid-phasesynthesis that is removable by the silylating agent is removed from thestarting peptide compound, the method comprising the step of contactingthe starting peptide compound with the silylating agent in a solvent.

In some aspects, the present invention relates to an amide compoundrepresented by formula (A) below or a salt thereof:

In formula (A), R_(1′) is selected from the group consisting ofhydrogen, Fmoc, Boc, Alloc, Cbz, Teoc, and trifluoroacetyl. R_(1′) ispreferably hydrogen, Fmoc, Boc, or Cbz.

In formula (A), R_(17A) and R_(17B) are both methyl, or R_(17A) andR_(17B), together with the nitrogen atom to which they are attached,form piperidine or morpholine.

In formula (A), R₁₈ is hydrogen or PG₁₀. PG₁₀ is selected from the groupconsisting of t-Bu, trityl, cumyl, benzyl, methyl, ethyl, allyl,optionally substituted aryl, optionally substituted aryl-C₁-C₄ alkyl,optionally substituted heteroaryl-C₁-C₄ alkyl, and2-(trimethylsilyl)ethyl. R₁₈ is preferably a hydrogen atom, t-Bu,benzyl, or allyl.

Preferred combinations of R_(1′) and R₁₈ in formula (A) include Fmoc anda hydrogen atom, Fmoc and allyl, Fmoc and t-Bu, Fmoc and benzyl, Boc anda hydrogen atom, Boc and allyl, Boc and t-Bu, Boc and benzyl, Cbz and ahydrogen atom, Cbz and allyl, Cbz and t-Bu, Cbz and benzyl, Alloc and ahydrogen atom, Alloc and allyl, Alloc and t-Bu, Alloc and benzyl, Teocand a hydrogen atom, Teoc and allyl, Teoc and t-Bu, Teoc and benzyl, ahydrogen atom and a hydrogen atom, a hydrogen atom and allyl, a hydrogenatom and t-Bu, and a hydrogen atom and benzyl.

Specific examples of the amide compound represented by formula (A)include the following compounds:

-   -   (1-1)        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-2) allyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-3) tert-butyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-4) benzyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-5)        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-6) allyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-7) tert-butyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-8) benzyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-9)        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-10) allyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-11) tert-butyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-12) benzyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-13)        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-14) allyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-15) tert-butyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-16) benzyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-17)        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic        acid,    -   (1-18) allyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-19) tert-butyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (1-20) benzyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (2-1)        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-2) allyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-3) tert-butyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-4) benzyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-5)        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-6) allyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-7) tert-butyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-8) benzyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-9)        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-10) allyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-11) tert-butyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-12) benzyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-13)        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-14) allyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-15) tert-butyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-16) benzyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-17)        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoic        acid,    -   (2-18) allyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-19) tert-butyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,    -   (2-20) benzyl        3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,    -   (3-1)        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-2) allyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-3) tert-butyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-4) benzyl        3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-5)        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-6) allyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-7) tert-butyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-8) benzyl        3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-9)        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-10) allyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-11) tert-butyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-12) benzyl        3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-13)        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoic        acid,    -   (3-14) allyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-15) tert-butyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-16) benzyl        3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,    -   (3-17)        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoic        acid,    -   (3-18) allyl        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,    -   (3-19) tert-butyl        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,    -   (3-20) benzyl        4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,    -   (4-1) 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoic acid,    -   (4-2) allyl 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (4-3) tert-butyl        3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (4-4) benzyl 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate,    -   (4-5) 3-(methylamino)-4-morpholino-4-oxobutanoic acid,    -   (4-6) allyl 3-(methylamino)-4-morpholino-4-oxobutanoate,    -   (4-7) tert-butyl 3-(methylamino)-4-morpholino-4-oxobutanoate,    -   (4-8) benzyl 3-(methylamino)-4-morpholino-4-oxobutanoate,    -   (4-9) 4-(dimethylamino)-3-(methylamino)-4-oxobutanoic acid,    -   (4-10) allyl 4-(dimethylamino)-3-(methylamino)-4-oxobutanoate,    -   (4-11) tert-butyl        4-(dimethylamino)-3-(methylamino)-4-oxobutanoate, or    -   (4-12) benzyl 4-(dimethylamino)-3-(methylamino)-4-oxobutanoate.

The amide compound of the present invention represented by formula (A)can be synthesized according to the following scheme, for example.

The amidation step in the above scheme can be achieved by condensing anyamine ((R_(17A))(R_(17B))NH) with a carboxyl group using a dehydrationcondensation agent such as a carbodiimide compound according toSolid-Phase Synthesis Handbook (publisher: Merck K.K., date ofpublication: May 1, 2002) or the method of Albert et al. (Synthesis,1987, 7, 635-637), for example.

The deprotection step in the above scheme can be achieved using themethod described in Greene's “Protective Groups in Organic Synthesis”(5th ed., John Wiley & Sons 2014) or the deprotection method describedherein, for example.

The compounds of the present invention may be either free forms or saltsthereof, both of which are included in the present invention. Examplesof such “salts” include inorganic acid salts, organic acid salts,inorganic base salts, organic base salts, and acidic or basic amino acidsalts.

Examples of the inorganic acid salts include hydrochlorides,hydrobromides, sulfates, nitrates, and phosphates. Examples of theorganic acid salts include acetates, succinates, fumarates, maleates,tartrates, citrates, lactates, stearates, benzoates, methanesulfonates,benzenesulfonates, and p-toluenesulfonates.

Examples of the inorganic base salts include alkali metal salts such assodium salts and potassium salts, alkaline earth metal salts such ascalcium salts and magnesium salts, aluminum salts, and ammonium salts.Examples of the organic base salts include diethylamine salts,diethanolamine salts, meglumine salts, and N,N-dibenzylethylenediaminesalts.

Examples of the acidic amino acid salts include aspartates andglutamates. Examples of the basic amino acid salts include argininesalts, lysine salts, and ornithine salts.

The compounds of the present invention may absorb water to form hydrateswhen left in the air, for example. Such hydrates are also included inthe present invention.

Further, the compounds of the present invention may absorb certain othersolvents to form solvates. Such solvates are also included in thepresent invention.

Herein, although structural formulas of the compounds of the presentinvention may represent certain isomers for convenience, the presentinvention includes all isomers and mixtures of isomers possible in termsof compound structure, such as geometric isomers, optical isomers, andtautomers, and the compounds are not limited to the formulas describedfor convenience. For example, when the compounds have an asymmetriccarbon atom in the molecule and exist as optically active isomers andracemates, both are included in the present invention.

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLE

The present invention will be further illustrated with reference to thefollowing Examples but is not limited thereto.

Solvents such as methylene chloride, ethyl acetate, 2-MeTHF,dichloroethane, or DMF used in the practice of the present inventionwere those from commercial suppliers, used without purification.Solvents such as dehydrated solvents, ultradehydrated solvents, oranhydrous solvents used for reactions without addition of water as asolvent were those from commercial suppliers, used without purification.

Unless otherwise stated, silyl compounds such as TMSOTf used in thepractice of the present invention, or reagents like electrophilicspecies scavengers such as BSA and BSTFA used in the practice of thepresent invention were those from commercial suppliers, used withoutpurification.

Unless otherwise stated, starting peptide or amide compounds used in thepractice of the present invention were those from commercial suppliers,used without purification. As necessary, such compounds were produced byknown methods and used.

In the following examples, analysis was performed by any one or more ofthe HPLC methods 1 to 4 or methods A, B, C, D, E, F, G, or H.

Analysis: HPLC (reaction conversion rate, purity)

HPLC Method 1

-   -   Instrument: Waters ACQUITY UPLC H-Class    -   Column: Ascentis Express C18 (2.7 μm, 4.6 mm×50 mm), Supelco    -   Eluent: A) 0.05% TFA/water, B) 0.05% TFA/CH₃CN    -   Gradient (B): 5% (0 min.)⇒100% (6 min.)⇒5% (7 min.)⇒5% (9 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm    -   Injection vol.: 5 μL    -   Sample prep.: 5 μL/1.00 mL

HPLC Method 2

-   -   Instrument: Waters LCT Premier    -   Column: Ascentis Express C18 (2.7 μm, 4.6 mm×50 mm), Supelco    -   Column temp.: 35 deg.    -   Eluent: A) 0.05% TFA/water, B) 0.05% TFA/CH3CN    -   Gradient (B): 50% (0 min.)⇒100% (6 to 11 min.)⇒50% (11 min.)⇒50%        (13 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm    -   Injection vol.: 5 μL    -   Sample prep.: 5 μL/1.00 mL

HPLC Method 3

-   -   Instrument: Waters ACQUITY UPLC H-Class    -   Column: Ascentis Express C18, (2.7 μm, 2.1 mm×50 mm), Supelco    -   Column temp.: 35 deg.    -   Eluent: A) 0.05% TFA/water, B) 0.05% TFA/CH3CN    -   Gradient (A): 95%(0 min)→0%(4.0 min)→0%(4.5 min)→95%(4.6        min)→95%(6 min)    -   Flow rate: 0.25 mL/min    -   Detection: PDA 210 nm (200-400 nm PDA total)    -   Injection vol.: 0.3 μL    -   Sample prep.: 50 μL/1.00 mL

HPLC Method 4

-   -   Instrument: Waters ACQUITY UPLC H-Class    -   Column: CAPCELL CORE ADME, (2.7 μm, 3.0 mm×150 mm)    -   Column temp.: 30 deg.    -   Solvents: A) 0.05% TFA/water, B) 0.05% TFA/CH3CN    -   Gradient (A): 70%(0 min)→30%(20.0 min)→0%(20.1 min)→0%(22.0        min)→70%(22.1 min)→70%(24 min)    -   Flow rate: 0.3 mL/min    -   Detection: PDA 254 nm (200-400 nm PDA total)    -   Injection vol.: 0.3 μL    -   Sample prep.: amorphous 10.0 mg in CH₃CN 10 ml; this solution        0.3 mL/0.7 mL CH₃CN

A. Boc Removal Reaction Experiments Example 1

Boc Removal Reaction of Boc-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 3a) (6-mer: TfOH-BSTFA Method)

77.7 mg of the raw material was weighed into a reaction vessel anddissolved in 5 v/w of ethyl acetate, and the reaction vessel was cooledwith ice. After 10 minutes, 0.055 ml (2.4 eq) of BSTFA and 3.0 μl (0.4eq) of TfOH were sequentially added under a nitrogen atmosphere, and thereaction solution was stirred. Three hours after the addition of thereagents, the reaction was analyzed by LCMS and the raw material wasconfirmed to disappear.

The reaction was quenched with saturated aqueous sodium bicarbonate. Theorganic layer was washed with saturated aqueous sodium bicarbonate and5% saline. The resulting organic layer was concentrated under reducedpressure to give 77.3 mg of a deprotected product quantitatively.

Amide bond cleavage was not confirmed.

TABLE 1 Weight (mg) Yield (%) Raw material (Compound 3a) 77.7 — Product(Compound 3b) 77.3 Quantitative

TABLE 2 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material906.55 807.55 5.976 93.83 (Compound 3a) ( [M − Boc + H]+) Product 806.49807.49 ( [M + H]+) 3.968 94.36 (Compound 3b)

Example 2

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-HMDS Method)

0.0810 g of the raw material was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate, and the reaction vessel was cooledwith ice. After 10 minutes, 0.079 ml (7.2 eq) of HMDS and 34 μl (3.6 eq)of TMSOTf were sequentially added under a nitrogen atmosphere, and thereaction solution was stirred. Four hours after the addition of thereagents, the reaction was analyzed by LCMS and the raw material wasconfirmed to disappear. Amide bond cleavage was not confirmed.

After confirming the disappearance of the raw material, the reactionsolution was stirred for another one hour and the reaction was analyzedby LCMS. Also at this time, amide bond cleavage was not confirmed.

To the reaction vessel was added 1 ml of brine to quench the reaction. 5ml of ethyl acetate was then added and the two layers were separated.The organic layer was washed with a mixture of 1 ml of saturated aqueoussodium bicarbonate and 1 ml of brine. The resulting organic layer wasconcentrated to give 68.0 mg of a deprotected product in 90% yield as atransparent film.

TABLE 3 Weight (mg) Yield (%) Raw material (Compound 1a) 81.0 — Product(Compound 1b) 68.0 90

TABLE 4 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) Purity MW m/z rt LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90 731.78 4.635 98.003

TABLE 5 Analysis (HPLC method 2: Mass spectroscopy) MW m/z rt Rawmaterial (Compound 1a) 1560.96 1561.4097 ( [M + H]+) 5.883 Product(Compound 1b) 1460.90 1461.5303 ( [M + H]+) 4.817

Example 3

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-HMDS Method)

The reaction was carried out according to Example 2. 19 hours after thereaction was started, the reaction was analyzed by LCMS. However,decomposition of the product was not confirmed.

TABLE 6 Weight (mg) Yield (%) Raw material (Compound 1a) 21.7 — Product(Compound 1b) Not isolated Not isolated

TABLE 7 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1241.89 6.476 98.408 Product (Compound 1b) 1460.90 731.76 4.562 97.256

Example 4

Boc Removal Reaction ofBoc-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-MeIle-Ala-MePhe-MeVal-Asp(OBn)-pip(Compound 2a) (11-mer: TfOH-BSTFA Method)

The reaction was carried out according to Example 1.

TABLE 9 Weight (mg) Yield (%) Raw material (Compound 2a) 48.1 — Product(Compound 2b) Not isolated Not isolated

TABLE 10 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 2a) 1610.97 ND 6.492 84.00 Product (Compound 2b) 1510.92 ND4.776 81.54

TABLE 11 Analysis (HPLC method 2: Mass spectroscopy) MW m/z rt Rawmaterial (Compound 2a) 1610.97 1611.43 ( [M + H]+) 5.96 Product(Compound 2b) 1510.92 1511.53 ( [M + H]+) 2.84

Example 5

Boc Removal Reaction of Boc-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 4a) (5-mer: TfOH-BSTFA Method)

The reaction was carried out according to Example 1.

TABLE 12 Weight (mg) Yield (%) Raw material (Compound 4a) 98.5 — Product(Compound 4b) 78.7 91

TABLE 13 Analysis (HPLC method 1) Purity LC MW m/z rt Area % Rawmaterial 821.49 844.4 ( [M + Na]+) 6.033 90.51 (Compound 4a) Product(Compound 4b) 721.44 722.46 ( [M + H]+) 3.879 93.30

Example 6

Reactions for Boc Removal of Boc-MeLeu-Thr(OBn)-MeGly-OAllyl (Compound8a) and its elongation to form Boc-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl (3mer: TfOH-BSTFA Method)

73.3 mg of the starting material was weighed into a reaction vessel anddissolved in 5 v/w of isopropyl acetate. After cooling the reactionvessel with ice, 0.085 ml of BSTFA and 0.0047 ml of TfOH were added andthe reaction solution was stirred for two hours. After confirming by LCthat deprotection was completed without main chain cleavage, 5 v/w ofwater and 186.9 mg of dipotassium hydrogenphosphate were added. 40.1 mgof Boc-Leu-OH monohydrate and 63.1 mg of DMT-MM were then added and thereaction solution was stirred with ice-cooling. 17 hours after theaddition of the reagents, completion of the reaction was confirmed byLC. The reaction was quenched by adding 5 v/w of a 1 N aqueous sodiumhydroxide solution. The organic layer was separated by liquid separationtreatment, then washed once with a 1 N aqueous sodium hydroxidesolution, twice with a 5% aqueous potassium bisulfate solution, and oncewith a 5% aqueous sodium chloride solution, and subsequentlyconcentrated under reduced pressure. 82.0 mg (93% yield) of a crudeproduct was obtained as an oily liquid.

TABLE 14 Weight (mg) Yield (%) Raw material (Compound 8a) 73.3 — Product(Compound 7a) 82.0 93

TABLE 15 Analysis of deprotection reaction (HPLC method 1) Purity MW m/zrt LC A % Raw material 547.33 448.30 5.513 96.17 (Compound 8a) ( [M −Boc + H]+) Boc-removed product 447.27 ND 2.938 95.95

TABLE 16 Analysis of elongation reaction (HPLC method 1) MW m/z rtPurity LC A % Product (Compound 8b) 660.85 561.28 5.657 95.36 ( [M −Boc + H]+)

Reactions for Teoc removal of Teoc-MeLeu-Thr(OBn)-MeGly-OAllyl (Compound80b) and its elongation to form Boc-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl (7a)(3-mer: TfOH-BSTFA Method)

Compound 7a can be obtained without main chain cleavage by removing theTeoc group in the same operation as in Example 6 and then reacting withBoc-Leu-OH.

Example 7

Boc Removal Reaction of Boc-MeIle-Ala-MePhe-MeVal-Asp(OBn)-pip (Compound5a) (5-mer: TfOH-BSTFA Method)

Deprotection and elongation reactions were performed in a one-potoperation according to the experimental method described in Example 6.

TABLE 17 Weight (mg) Yield (%) Raw material (Compound 5a) 105.3 —Product (Compound 2a) 153.6 78%

TABLE 18 Analysis of deprotection reaction (HPLC method 1) MW m/z rtPurity LC A % Raw material 862.52 573.33 5.978 95.01 (Compound 5a)Deprotected 762.47 763.47 ([M + H]+) 3.779 94.05 product (Compound 5b)

TABLE 19 Analysis of the product after elongation (HPLC method 1:Determination of purities of the raw material and target product) MW m/zrt Purity LC A % Product (Compound 2a) 1610.97 ND 6.699 85.93

TABLE 20 Analysis of the product after elongation (HPLC method 2: Massspectroscopy) MW m/z rt Product 1610.97 1611.43 ([M + H]+) 5.96(Compound 2a)

Example 8

Boc Removal and Elongation Reactions of Boc-Ala-MePhe-MeVal-Asp(OBn)-pip(Compound 6a) (4-mer: TfOH-BSTFA Method)

Deprotection and elongation reactions were performed in a one-potoperation according to the experimental method described in Example 6.

TABLE 21 Weight (mg) Yield (%) Raw material (Compound 6a) 277 — Product(Compound 5a) 308.9 95%

TABLE 22 Analysis of the deprotected product (HPLC method 1) MW m/z rtPurity LC A % Raw material 735.42  758.4 ([M + Na]+) 5.357 98.921(Compound 6a) Deprotected 635.37 636.40 ([M + H]+) 3.383 97.893 product(Compound 6b)

TABLE 23 Analysis after elongation reaction (HPLC method 1) MW m/z rtPurity LC A % Product (Compound 5a) 862.52 573.33 5.978 96.938

Example 9

Boc Removal Reaction of Boc-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl (Compound7a) (4-mer: TfOH-BSTFA Method)

Deprotection and elongation reactions were performed in a one-potoperation according to the experimental method described in Example 6.

TABLE 24 Weight (mg) Yield (%) Raw material (Compound 7a) 82.0 — Product(Compound 4a) 98.5 97

TABLE 25 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material660.41 561.28 ([M − 5.657 95.36 (Compound 7a) 100 + H]+) Intermediate560.36 561.40 ([M + H]+) 3.545 94.04 (Compound 7b)

TABLE 26 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product 821.49844.4 ([M + Na]+) 6.034 90.36 (Compound 4a)

Example 10

Boc Removal Reaction of Boc-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl (Compound7a) Under Known Conditions (4-Mer: HCl Method) (Russ. J. Bioorg. Chem.,2016, 42, 143.)

18.7 mg of the raw material was weighed into a reaction vessel anddissolved in 10 v/w of trifluoroethanol. 8.5 μl (1.2 eq) of 4 N HCl(ethyl acetate solution) was then added and the reaction solution wasstirred. Two hours after the addition of the reagent, the reaction wasanalyzed by LCMS (HPLC method 1) and the conversion rate (=targetproduct/(target product+starting material)) was confirmed to be 81%. Atthis time, 0.861% of H-Thr(OBn)-MeGly-OAllyl (Compound 7c) and 1.275% ofH-Leu-MeLeu-Thr(OBn)-OH (Compound 7d) were respectively detected ascompounds in which amide bonds were cleaved.

TABLE 27 Analysis (HPLC method 1) MW m/z rt LC A % Raw material 660.41561.28 ([M − 5.657 18.437 (Compound 7a) Boc + H]+) Product 560.36 561.40([M + H]+) 3.363 79.258 (Compound 7b) Cleaved 320.17 321.08 ([M + H]+)2.500 0.861% product (Compound 7c) Cleaved 449.29 450.17 ([M + H]+)2.759 1.275% product (Compound 7d)

Example 11

Reactions for Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)and its Elongation to Form Boc-Ala-MePhe-MeVal-Asp(OBn)-pip (3-mer:TMSOTf-BSTFA Method)

Deprotection and elongation reactions were performed in a one-potoperation according to the experimental method described in Example 6.

TABLE 28 Weight (mg) Yield (%) Raw material (Compound 9a) 253.2 —Product (Compound 6a) 319.4 Quantitative

TABLE 29 Analysis of deprotection reaction (HPLC method 1) MW m/z rtPurity LC A % Raw material 664.38  687.5 ([M + Na]+) 5.647 99.206(Compound 9a) Deprotected 564.33 565.31 ([M + H]+) 3.338 98.885 product

TABLE 30 Analysis of elongation reaction (HPLC method 1) MW m/z rtPurity LC A % Product 735.92 758.4 ([M + Na]+) 5.377 99.538 (Compound6a)

Example 12

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a) (3-Mer:TMSOTf-MSTFA Method)

Deprotection and elongation reactions were performed in a one-potoperation according to the experimental method described in Example 6.

TABLE 31 Weight (mg) Yield (%) Raw material (Compound 9a) 200.6 —Product (Compound 6a) 220.6 95

TABLE 32 Analysis of deprotection reaction (HPLC method 1) MW m/z rtPurity LC A % Raw material 664.38  687.5 ([M + Na]+) 5.647 99.206(Compound 9a) Deprotected 564.33 565.31 ([M + H]+) 3.338 97.921 product(Compound 9b)

TABLE 33 Analysis of elongation reaction (HPLC method 1) MW m/z rtPurity LC A % Product 735.92 758.4 ([M + Na]+) 5.308 99.279 (Compound6a)

Example 13

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a) (3-mer:TMSOTf-BSA Method, TMSOTf 2.4 Eq, BSA 2.4 Eq)

Deprotection and elongation reactions were performed in a one-potoperation according to the experimental method described in Example 6.

TABLE 34 Weight (mg) Yield (%) Raw material (Compound 9a) 204.6 —Product (Compound 6a) 219.0 97

TABLE 35 Analysis of deprotection reaction (HPLC method 1) MW m/z rtPurity LC A % Raw material 664.38  687.5 ([M + Na]+) 5.647 99.206(Compound 9a) Deprotected 564.33 565.31 ([M + H]+) 3.338 98.970 product(Compound 9b)

TABLE 36 Analysis of elongation reaction (HPLC method 1) MW m/z rtPurity LC A % Product 735.92 758.5 ([M + Na]+) 5.301 99.573 (Compound6a)

Example 14

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a) (3-mer:TMSOTf-HMDS Method)

107.4 mg of the raw material was weighed into a reaction vessel anddissolved in 5 v/w of ethyl acetate, and the reaction vessel was cooledwith ice. After 10 minutes, 0.085 ml (2.4 eq) of HMDS(1,1,1,3,3,3-hexamethyldisilazane) and 35 μl (1.2 eq) of TMSOTf weresequentially added under a nitrogen atmosphere, and the reactionsolution was stirred. Two hours after the addition of the reagents, thereaction was analyzed by LCMS to find that the conversion rate was55.8%. Therefore, 20 μl (0.69 eq) of TMOSTf was further added. One hourafter the further addition of the reagent, the raw material wasconfirmed to disappear by LCMS. Amide bond cleavage was not confirmed.

To the reaction vessel was added 373.4 mg (13.3 eq) of dipotassiumhydrogenphosphate, after which 0.89 ml of water was added and thereaction solution was stirred for 20 minutes.

To the reaction vessel was then added 3 ml of ethyl acetate, and the twolayers were separated. The organic layer was washed with a mixture of 1ml of saturated aqueous sodium bicarbonate and 1 ml of brine. Theresulting organic layer was concentrated.

The resulting oily liquid was then dissolved in 1 ml of ethyl acetate,and 251.0 mg of dipotassium hydrogenphosphate was added. After adding0.89 ml of water, the reaction vessel was cooled with ice. 36.8 mg ofBoc-Ala-OH and 79.5 mg DMT-MM were added, and the reaction solution wasstirred overnight in an ice bath. After 15.5 hours, the raw material wasconfirmed to disappear by LCMS and liquid separation treatment was thenconducted. The resulting organic layer was washed twice with 5%potassium carbonate, once with water, twice with 5% potassium bisulfate,and once with brine, and concentrated. 0.1075 g (90% yield) of a crudeproduct was obtained as a white solid.

TABLE 37 Weight (mg) Yield (%) Raw material (Compound 9a) 107.4 —Product (Compound 6a) 107.5 90

TABLE 38 Analysis of deprotection reaction (HPLC method 1) MW m/z rtPurity LC A % Raw material 664.38  687.5 ([M + Na]+) 5.647 99.206(Compound 9a) Intermediate 564.33 565.31 ([M + H]+) 3.360 98.783(Compound 9b)

TABLE 39 Analysis of elongation reaction (HPLC method 1) MW m/z rtPurity LC A % Product 735.42 758.4 ([M + Na]+) 5.268 98.031 (Compound9a)B. Screening of electrophilic species scavengers

Example 15

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a) (Base:N-(Trimethylsilyl)diethylamine)

99.5 mg of the raw material was weighed into a reaction vessel anddissolved in 5 v/w of ethyl acetate, and the reaction vessel was cooledwith ice. 0.070 ml (2.4 eq) of N-((trimethylsilyl)diethylamine) and0.032 ml (1.2 eq) of TMSOTf were sequentially added under a nitrogenatmosphere, and the reaction solution was stirred. 1.5 hours after theaddition of the reagents, the reaction was analyzed by LCMS to find thatthe conversion rate was 30%. Therefore, 0.029 ml (1.1 eq) of TMOSTf wasfurther added. Four hours after the start of the reaction, the reactionwas analyzed by LCMS and the raw material was confirmed to disappear. Atthis time, amide bond cleavage was not confirmed. The reaction solutionwas stirred overnight, and 20 hours after the start of the reaction, thereaction was analyzed again by LCMS. Also at this time, amide bondcleavage was not confirmed. 21 hours after the start of the reaction,0.2032 g of dipotassium hydrogenphosphate and 0.50 mL of water wereadded to the reaction solution which was then stirred for 30 min.

The reaction solution was diluted with 4 ml of ethyl acetate, and thetwo layers were then separated to provide an organic layer. Theresulting organic layer was washed twice with a mixed solvent of 0.5 mlof brine and 0.5 ml of saturated aqueous sodium bicarbonate. The organiclayer was then washed with 0.5 ml of brine and concentrated. The residuewas dissolved in 4 ml of isopropyl acetate and washed twice with a mixedsolvent of 1 ml of 0.5 M aqueous sodium hydroxide and 0.5 ml of brine.Then, the organic layer was washed with 0.5 ml of 10% saline andconcentrated under reduced pressure to give 81.7 mg of a deprotectedproduct in 97% yield.

TABLE 40 Weight (mg) Yield (%) Raw material (Compound 9a) 99.5 — Product(Compound 9b) 81.7 97

TABLE 41 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.27 ([M + H]+) 3.364 95.547 (Compound 9b)

Example 16

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: N-(Trimethylsilyl)morpholine)

The experiment was performed according to the method described inExample 15.

TABLE 42 Weight (mg) Yield (%) Raw material (Compound 9a) 98.4 — Product(Compound 9b) 117.8 Quantitative

TABLE 43 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.30 ([M + H]+) 3.350 98.652 (Compound 9b)

Example 17

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: N-tert-Butyltrimethylsilylamine)

The experiment was performed according to the method described inExample 15.

TABLE 44 Weight (mg) Yield (%) Raw material (Compound 9a) 100.9 —Product (Compound 9b) 128.6 Quantitative

TABLE 45 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.28 ([M + H]+) 3.354 98.631 (Compound 9b)

Example 18

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: 2,2,4,4-Tetramethylpentanoneimine)

The experiment was performed according to the method described inExample 15.

TABLE 46 Weight (mg) Yield (%) Raw material (Compound 9a) 88.0 — Product(Compound 9b) 102.5 Quantitative

TABLE 47 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.28 ([M + H]+) 3.338 98.433 (Compound 9b)

Example 19

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: Isopropenyloxytrimethylsilane)

The experiment was performed according to the method described inExample 15.

TABLE 48 Weight (mg) Yield (%) Raw material (Compound 9a) 44.3 — Product(Compound 9b) 44.2 Quantitative

TABLE 49 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.27 ([M + H]+) 3.340 97.587 (Compound 9b)

Example 20

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: Dimethylketene methyl trimethylsilyl acetal)

The experiment was performed according to the method described inExample 15.

TABLE 50 Weight (mg) Yield (%) Raw material (Compound 9a) 44.9 — Product(Compound 9b) 44.7 Quantitative

TABLE 51 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.27 ([M + H]+) 3.348 98.352 (Compound 9b)

Example 21

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: 3,4-Dihydro-2H-pyran)

The experiment was performed according to the method described inExample 15.

TABLE 52 Weight (mg) Yield (%) Raw material (Compound 9a) 53.7 — Product(Compound 9b) 69.2 Quantitative

TABLE 53 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.27 ([M + H]+) 3.325 98.503 (Compound 9b)

Example 22

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: N,N,N′,N′-Tetramethyl-1,8-naphthalenediamine)

The experiment was performed according to the method described inExample 15.

TABLE 54 Weight (mg) Yield (%) Raw material (Compound 9a) 50.6 — Product(Compound 9b) 57.6 Quantitative

TABLE 55 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material664.38  687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33565.28 ([M + H]+) 3.353 98.409 (Compound 9b)

(LCA % of the product described above is a value obtained by excludingthe base remaining after post-treatment.)

Example 23

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: 2,2′-Isopropylidenebis[(4S)-4-tert-butyl-2-oxazoline])

The experiment was performed according to the method described inExample 15.

TABLE 56 Weight (mg) Yield (%) Raw material (Compound 9a) 45.7 — Product(Compound 9b) 128.5 Quantitative

TABLE 57 Analysis (HPLC method 1) MW m/z rt LC A % Raw material 664.38 687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33 565.28([M + H]+) 3.353 90.099 (Compound 9b)

(LCA % of the product described above is a value obtained by excludingthe base remaining after post-treatment.)

Example 24

Deprotection of Boc-MePhe-MeVal-Asp(OBn)-pip (Compound 9a)

(Base: Diisopropylcarbodiimide)

The experiment was performed according to the method described inExample 15.

TABLE 58 Weight (mg) Yield (%) Raw material (Compound 9a) 36.8 — Product(Compound 9b) 31.2 85

TABLE 59 Analysis (HPLC method 1) MW m/z rt LC A % Raw material 664.38 687.5 ([M + Na]+) 5.647 99.206 (Compound 9a) Product 564.33 565.28([M + H]+) 3.347 89.920 (Compound 9b)

(LCA % of the product described above is a value obtained by excludingthe base remaining after post-treatment.)

C. Screening of Electrophilic Species Scavengers (Experiments in 11-MerPeptides) Example 25

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-BSTFA Method)

The experiment was performed according to Example 2.

TABLE 60 Weight (mg) Yield (%) Raw material (Compound 1a) 19.7 — Product(Compound 1b) 14.8 80

TABLE 61 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.67 4.638 96.275

Example 26

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-BSA Method)

The experiment was performed according to Example 2.

TABLE 62 Weight (mg) Yield (%) Raw material (Compound 1a) 18.0 — Product(Compound 1b) 11.8 70

TABLE 63 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.67 4.633 94.636

Example 27

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-silylamine Method)

The experiment was performed according to Example 2.

TABLE 64 Weight (mg) Yield (%) Raw material (Compound 1a) 19.3 — Product(Compound 1b) 16.1 89

TABLE 65 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.68 4.627 96.267

Example 28

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-silylamine Method)

The experiment was performed according to Example 2.

TABLE 66 Weight (mg) Yield (%) Raw material (Compound 1a) 20.2 — Product(Compound 1b) 15.3 81

TABLE 67 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.65 4.626 97.574

Example 29

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-imine Method)

The experiment was performed according to Example 2.

TABLE 68 Weight (mg) Yield (%) Raw material (Compound 1a) 18.7 — Product(Compound 1b) 15.7 90

TABLE 69 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.70 4.631 97.089

Example 30

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-MSTFA Method)

The experiment was performed according to Example 2.

TABLE 70 Weight (mg) Yield (%) Raw material (Compound 1a) 21.7 — Product(Compound 1b) 15.7 77

TABLE 71 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.61 4.616 96.490

Example 31

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-silylamine Method)

The experiment was performed according to Example 2.

TABLE 72 Weight (mg) Yield (%) Raw material (Compound 1a) 17.5 — Product(Compound 1b) 13.5 81

TABLE 73 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.71 4.629 97.390

Example 32

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-ketene silyl acetal Method)

The experiment was performed according to Example 2.

TABLE 74 Weight (mg) Yield (%) Raw material (Compound 1a) 23.6 — Product(Compound 1b) 19.8 90

TABLE 75 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.63 4.620 97.488

Example 33

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-Proton Sponge Method)

The experiment was performed according to Example 2.

TABLE 76 Weight (mg) Yield (%) Raw material (Compound 1a) 17.5 — Product(Compound 1b) 16.2 97

(The base remains even after post-treatment.)

TABLE 77 Analysis (HPLC method 1: Determination of purities of the rawmaterial and target product) MW m/z rt Purity LC A % Raw material(Compound 1a) 1560.96 1242.05 6.519 96.680 Product (Compound 1b) 1460.90731.68 4.628 96.025

(LCA % of the product described above is a value obtained by excludingthe base remaining after post-treatment.)

Example 34

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-lutidine Method)

18.8 mg of the raw material was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. 0.005 ml (3.6 eq) of 2,6-lutidineand 0.0078 ml (3.6 eq) of TMSOTf were then added and the reactionsolution was stirred. Four hours after the addition of the reagents, thereaction was analyzed by LCMS (HPLC method 1) and the conversion rate(=target product/(target product+starting material)) was confirmed to be14%. Next, the reaction was analyzed by LCMS 19 hours after the additionof the reagents to find that the conversion rate was still 14%. Amidebond cleavage was not confirmed.

TABLE 78 Weight (mg) Yield (%) Raw material (Compound 1a) 18.8 — Product(Compound 1b) Not isolated Not isolated

TABLE 79 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.519 96.680

TABLE 80 Analysis (HPLC method 1: Determination of conversion rate) MWm/z rt LC A % Raw material (Compound 1a) 1560.96 1242.05 6.519 75.059Product (Compound 1b) 1460.90 731.67 4.620 11.831

Example 35

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-lutidine Method)

89.3 mg of the raw material was weighed into a reaction vessel anddissolved in 1 ml of ethyl acetate. 0.016 ml (2.4 eq) of 2,6-lutidineand 0.012 ml (1.2 eq) of TMSOTf were then added and the reactionsolution was stirred. 1.25 hours after the addition of the reagents, thereaction was analyzed by LCMS (HPLC method 1) to find that theconversion rate (=target product/(target product+starting material)) was0%. Therefore, 0.032 ml (4.8 eq) of 2,6-lutidine and 0.024 ml (2.4 eq)of TMSOTf were further added. The reaction was analyzed by LCMS, 1.25hours and 14 hours after the further addition of the reagents,respectively, to find that the conversion rate was still 0%. Amide bondcleavage was not confirmed.

TABLE 81 Weight (mg) Yield (%) Raw material (Compound 1a) 89.3 — Product(Compound 1b) Not isolated Not isolated

TABLE 82 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.545 89.564

TABLE 83 Analysis (HPLC method 1: Determination of conversion rate) MWm/z rt LC A % Raw material (Compound 1a) 1560.96 1242.05 6.571 89.649Product (Compound 1b) 1460.90 ND ND 0

Example 36

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-2,6-di-tert-butylpyridine Method)

14.9 mg of the raw material was weighed into a reaction vessel anddissolved in 0.2 ml of ethyl acetate. 0.011 ml (5.4 eq) of2,6-di-t-butylpyridine and 0.0062 ml (3.6 eq) of TMSOTf were then addedand the reaction solution was stirred. Four hours after the addition ofthe reagents, the reaction was analyzed by LCMS (HPLC method 1) to findthat the conversion rate (=target product/(target product+startingmaterial)) was 59%. The reaction was analyzed again by LCMS 22 hoursafter the start of the reaction to find that the conversion rate wasstill 59%. Amide bond cleavage was not confirmed.

TABLE 84 Weight (mg) Yield (%) Raw material (Compound 1a) 14.9 — Product(Compound 1b) Not isolated Not isolated

TABLE 85 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.545 96.680

TABLE 86 Analysis (HPLC method 1: Determination of conversion rate) MWm/z rt LC A % Raw material (Compound 1a) 1560.96 1242.05 6.571 26.863Product (Compound 1b) 1460.90 731.67 4.620 38.429 Ethyl acetate 1.85012.893 2,6-di(tBu)pyridine 3.844 19.629

Example 37

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-tert-amine Method)

17.7 mg of the raw material was weighed into a reaction vessel anddissolved in 0.20 ml of ethyl acetate. 0.0057 ml (3.6 eq) oftriethylamine and 0.0074 ml (3.6 eq) of TMSOTf were then added and thereaction solution was stirred. The reaction was analyzed by LCMS (HPLCmethod 1) one hour and four hours after the addition of the reagents tofind that the conversion rate (=target product/(target product+startingmaterial)) was merely 0.7% at each time. Amide bond cleavage was notconfirmed.

TABLE 87 Weight (mg) Yield (%) Raw material (Compound 1a) 17.7 — Product(Compound 1b) Not isolated Not isolated

TABLE 88 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.519 96.680

TABLE 89 Analysis (HPLC method 1: Determination of conversion rate) MWm/z rt LC A % Raw material (Compound 1a) 1560.96 1242.05 6.579 84.391Product (Compound 1b) 1460.90 731.67 4.639 0.599 Ethyl acetate (Solvent)1.844 13.474

Example 38

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-tert-amine Method)

19.1 mg of the raw material was weighed into a reaction vessel anddissolved in 0.20 ml of ethyl acetate. 0.0074 ml (3.6 eq) ofdiisopropylethylamine and 0.0077 ml (3.6 eq) of TMSOTf were then addedand the reaction solution was stirred. The reaction was analyzed by LCMS(HPLC method 2) two hours and four hours after the addition of thereagents to find that the conversion rate (=target product/(targetproduct+starting material)) was merely 0.8% or less at each time. Amidebond cleavage was not confirmed.

TABLE 90 Weight (mg) Yield (%) Raw material (Compound 1a) 19.1 — Product(Compound 1b) Not isolated Not isolated

TABLE 91 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.519 96.680

TABLE 92 Analysis (HPLC method 1: Determination of conversion rate) MWm/z rt LC A % Raw material (Compound 1a) 1560.96 1241.79 6.568 85.820Product (Compound 1b) 1460.90 731.68 4.629 0.458 Ethyl acetate (Solvent)1.836 12.495

Example 39

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TMSOTf-tert-amine Method)

92.9 mg of the raw material was weighed into a reaction vessel anddissolved in 1 ml of ethyl acetate, and the reaction vessel was thencooled with ice. 0.015 ml (2.4 eq) of diisopropylethylamine and 0.013 ml(1.2 eq) of TMSOTf were then added under a nitrogen atmosphere, and thereaction solution was stirred. The reaction was analyzed two hours afterthe addition of the reagents to find that the conversion rate (=targetproduct/(target product+starting material)) was 0%. Therefore, 0.030 ml(4.8 eq) of diisopropylethylamine and 0.026 ml (2.4 eq) of TMSOTf werefurther added three hours after the addition of the reagents. Threehours after the further addition of the reagents, the reaction wasanalyzed again to find that the conversion rate was 0%. Amide bondcleavage was not confirmed.

TABLE 93 Weight (mg) Yield (%) Raw material (Compound 1a) 92.9 — Product(Compound 1b) Not isolated Not isolated

TABLE 94 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961241.93 6.538 89.465

TABLE 95 Analysis (HPLC method 1: Determination of conversion rate) MWm/z rt LC A % Raw material (Compound 1a) 1560.96 1242.05 6.568 89.432Product (Compound 1b) 1460.90 ND ND 0

Example 40

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: conducted using only TMSOTf in the absence of anorganic base.)

18.3 mg of the raw material was weighed into a reaction vessel anddissolved in 0.2 ml of ethyl acetate, and the reaction vessel was thencooled with ice. 0.0077 ml (3.6 eq) of TMSOTf was then added under anitrogen atmosphere, and the reaction solution was stirred. The reactionwas analyzed 1.5 hours after the addition of the reagent to confirm thatmultiple amide bond-cleaved products were produced at a conversion rate(=target product/(target product+starting material)) of 68%.

TABLE 96 Weight (mg) Yield (%) Raw material (Compound 1a) 18.3 — Product(Compound 1b) Not isolated Not isolated

TABLE 97 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.519 96.680

TABLE 98 Analysis (HPLC method 1: Determination of conversion rate andrates of production of by-products) MW m/z rt LC A % Raw material(Compound 1a) 1560.96 1241.77 6.575 15.543 Product (Compound 1b) 1460.90731.72 4.630 32.712 Cleaved product (Compound 1c) 377.23 378.22 ([M +H]+) 2.269 0.986 Cleaved product (Compound 1d) 757.47 758.40 ([M + H]+)2.758 9.731 Cleaved product (Compound 1e) 721.44 722.43 ([M + H]+) 3.89315.859 Cleaved product (Compound 1f) 1101.68 1102.56 ([M + H]+)  4.2204.701 Cleaved product (Compound 1g) Unknown 573.27 4.788 4.808 Ethylacetate 1.840 15.660The structure of Compound 1g is unknown.

Example 41

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: conducted with TMSOTf and triisopropylsilane inthe absence of an organic base.)

20.5 mg of the raw material was weighed into a reaction vessel anddissolved in 0.2 ml of ethyl acetate, and the reaction vessel was thencooled with ice. 0.0097 ml (3.6 eq) of triisopropylsilane and 0.0085 ml(3.6 eq) of TMSOTf were then added under a nitrogen atmosphere, and thereaction solution was stirred. The reaction was analyzed one hour afterthe addition of the reagents to confirm that multiple e bond-cleavedproducts were produced at a conversion rate (=target product/(targetproduct+starting material)) of 43%.

TABLE 99 Weight (mg) Yield (%) Raw material (Compound 1a) 20.5 — Product(Compound 1b) Not isolated Not isolated

TABLE 100 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.538 96.680

TABLE 101 Analysis (HPLC method 1: Determination of conversion rate andrates of production of by- products) MW m/z rt LC A % Raw material(Compound 1a) 1560.96 1241.77 6.575 28.216 Product (Compound 1b) 1460.90731.59 4.624 20.995 Cleaved product (Compound 1c) 377.23 378.24 ([M +H]+) 2.259 0.479 Cleaved product (Compound 1d) 757.47 758.44 ([M + H]+)2.750 5.875 Cleaved product (Compound 1e) 721.44 722.36 ([M + H]+) 3.88515.802 Cleaved product (Compound 1f) 1101.68 1102.55 ([M + H]+)  4.2125.239 Impurity (Compound 1g) Unknown 573.24 4.780 8.178 Ethyl acetate1.836 14.570The structure of Compound 1g is unknown.

Example 42

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TFA Method)

35.3 mg of the raw material was weighed into a reaction vessel anddissolved in 10 v/w of methylene chloride, and the reaction vessel wasthen cooled with ice. 0.004 ml (2.3 eq) of trifluoroacetic acid was thenadded under a nitrogen atmosphere, and the reaction solution wasstirred. The reaction was analyzed 10 minutes after the addition of thereagent to find that the conversion rate (=target product/(targetproduct+starting material)) was 0%. Therefore, 0.004 ml (2.3 eq) oftrifluoroacetic acid was further added. The reaction was analyzed again10 minutes after the further addition of the reagent to find that theconversion rate was 0%. Therefore, 0.0094 ml (5.4 eq) of trifluoroaceticacid was further added. 10 minutes after the further addition of thereagent, the reaction solution was warmed to room temperature andcontinued to be stirred. Four hours after warming to room temperature,the reaction was analyzed to confirm that an amide bond-cleaved product1e was produced at a conversion rate of 2%.

TABLE 102 Weight (mg) Yield (%) Raw material (Compound 1a) 35.3 —Product (Compound 1b) Not isolated Not isolated

TABLE 103 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.519 96.680

TABLE 104 Analysis (HPLC method 1: Determination of conversion rate andrates of production of by-products) MW m/z rt LC A % Raw material(Compound 1a) 1560.96 1242.05 6.568 93.488 Product (Compound 1b) 1460.90731.67 4.637 2.048 Cleaved product (Compound 1e) 721.44 722.47 ([M +H]+) 3.900 0.880 Impurity (Compound 1g) Unknown 573.29 4.789 1.020Methylene chloride 2.375 1.059The structure of Compound 1g is unknown.

Example 43

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TFA-TIPS-H₂O-PhOH Method)

Reference: J. Am. Chem. Soc., 2015, 137, 13488

15.9 mg of the raw material was weighed into a reaction vessel, 0.0125ml of water and 0.0125 ml of triisopropylsilane were added, and thereaction vessel was cooled to −10° C. 0.48 ml of trifluoroacetic acidwas then added and the reaction solution was stirred. The reaction wasanalyzed two hours after the addition of the reagent to confirm thatamide bond-cleaved products 1d and 1e were produced at a conversion rate(=target product/(target product+starting material)) of 89%. Thereaction was analyzed again 14.5 hours after the addition of the reagentto confirm that the raw material was consumed and the amide bond-cleavedproducts 1d and 1e were increasingly produced.

TABLE 105 Weight (mg) Yield (%) Raw material (Compound 1a) 15.9 —Product (Compound 1b) Not isolated Not isolated

TABLE 106 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.519 96.680

TABLE 107 Analysis (HPLC method 1: Determination of conversion rate andrates of production of by-products at two hours after the reaction) LCMW m/z rt A % Raw material 1560.96 1241.57 6.527 9.531 (Compound 1a)Product (Compound 1b) 1460.90 731.67 4.629 77.361 Cleaved product 757.47758.41 ([M + H]+) 2.761 5.716 (Compound 1d) Impurity (Compound 1e)721.44 722.47 ([M + H]+) 3.902 7.392

TABLE 108 Analysis (HPLC method 1: Rates of production of the targetproduct and by-products at 14.5 hours after the reaction) LC MW m/z rt A% Raw material 1560.96 ND ND 0 (Compound 1a) Product (Compound 1b)1460.90 731.67 4.631 29.383 Cleaved product 757.47 758.39 ([M + H]+)2.757 37.117 (Compound 1d) Cleaved product 721.44 722.41 ([M + H]+)3.892 32.950 (Compound 1e)

Example 44

Boc Removal Reaction ofBoc-MeIle-Ala-MePhe-MeVal-Asp(pip)-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl(Compound 1a) (11-mer: TFA-TIPS-H₂O-PhOH Method) Reference: J. Am. Chem.Soc., 2012, 134, 13244

10.3 mg of phenol was weighed into a reaction vessel, 0.034 ml of waterand 0.025 ml of triisopropylsilane were added, and the reaction vesselwas cooled to −10° C. 0.50 ml of trifluoroacetic acid was then added.Six minutes after the addition, 13.4 mg of the raw material was addedand the reaction solution was stirred. The reaction was analyzed twohours after the addition of the raw material to confirm that amidebond-cleaved products 1d and 1e were produced at a conversion rate(=target product/(target product+starting material)) of 82%. Thereaction was analyzed again seven hours after the addition of the rawmaterial to confirm that the raw material was consumed and the amidebond-cleaved products 1d and 1e were increasingly produced.

TABLE 109 Weight (mg) Yield (%) Raw material (Compound 1a) 10.3 —Product (Compound 1b) Not isolated Not isolated

TABLE 110 Analysis (HPLC method 1: Determination of purity of the rawmaterial) MW m/z rt Purity LC A % Raw material (Compound 1a) 1560.961242.05 6.538 96.680

TABLE 111 Analysis (HPLC method 1: Determination of conversion rate andrates of production of by-products at two hours after the reaction) LCMW m/z rt A % Raw material 1560.96 1242.05 6.523 6.500 (Compound 1a)Product (Compound 1b) 1460.90 731.67 4.628 30.640 Cleaved product 757.47758.38 ([M + H]+) 2.769 1.583 (Compound 1d) Cleaved product 721.44722.37 ([M + H]+) 3.884 2.659 (Compound 1e) Phenol 2.172 58.619

TABLE 112 Analysis (HPLC method 1: Rates of production of the targetproduct and by-products at five hours after the reaction) LC MW m/z rt A% Raw material 1560.96 ND ND 0 (Compound 1a) Product (Compound 1b)1460.90 731.67 4.633 25.258 Cleaved product 757.47 758.40 ([M + H]+)2.769 8.506 (Compound 1d) Cleaved product 721.44 722.39 ([M + H]+) 3.8848.035 (Compound 1e) Phenol 2.172 58.200

As shown in Examples 2, 3, and 26 to 44, it was found that imidates,amides, ketene acetals, enol ethers, imines, amines, diamines, anddialkylcarbodiimides are excellent as electrophilic species scavengers,suppress main chain damage, and allow efficient progress of Boc removalreaction of peptides having a long main chain such as 11-mers.

D. Experiment of Comparison Between the TFA Method and the TMSOTf-HMDSMethod in tBu Removal Reactions (Table 113)

Example 45

t-Bu Removal Reaction of Compound 13a (TFA Method)

29.8 mg of Substrate 13a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA(10 eq.) at room temperature. Four hours after the addition of thereagent, TFA (10 eq.) was further added. 1.5 hours after the furtheraddition of the reagent, the reaction was analyzed by LCMS to find thatthe reaction conversion rate was 52%. The purity was reduced by 22%,while 10% of a by-product due to amide bond cleavage was observed.

Example 46

t-Bu Removal Reaction of Compound 13a (TMSOTf-HMDS Method)

30.0 mg of Substrate 13a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (3.0 eq.) and TMSOTf (2.0 eq)were sequentially added to the solution at 0° C. Two hours after theaddition of the reagents, the reaction solution was warmed to roomtemperature. 2.5 hours after warming, HMDS (3 eq.) and TMSOTf (2 eq.)were further added. 30 minutes after the further addition of thereagents, the reaction was analyzed by LCMS to confirm that theconversion rate was 99% or more. At this time, amide bond cleavage wasnot confirmed.

Example 47

t-Bu Removal Reaction of Compound 14a (TFA Method)

29.7 mg of Substrate 14a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA(20 eq.) at room temperature. Seven hours after the addition of thereagent, the reaction was analyzed by LCMS to find that the reactionconversion rate was 92%. The purity was reduced by 46%, while 24% of aby-product due to amide bond cleavage was observed.

Example 48

t-Bu Removal Reaction of Compound 14a (TMSOTf-HMDS Method)

30.0 mg of Substrate 14a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (3.0 eq.) and TMSOTf (2.0 eq)were sequentially added to the solution at 0° C. Six hours after theaddition of the reagents, the reaction was analyzed by LCMS and the rawmaterial was confirmed to disappear. At this time, amide bond cleavagewas not confirmed. After confirming the completion of the reaction, 44.5mg (8.0 eq) of potassium hydrogenphosphate and 0.30 mL of water wereadded to the reaction solution, which was then stirred for 30 minuteswith ice-cooling. 1 mL of ethyl acetate was added to the reactionsolution, which was then washed with 0.30 mL of brine. The organic layerwas filtered through celite, then concentrated, and dried under reducedpressure to give 26.8 mg of 2b in a purity of 99%.

Example 49

t-Bu Removal Reaction of Compound 15a (TFA Method)

29.9 mg of Substrate 15a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA(20 eq.) at room temperature. 5.5 hours after the addition of thereagent, the reaction was analyzed by LCMS to find that the reactionconversion rate was 50%. 28% of a by-product due to amide bond cleavagewas observed.

Example 50

t-Bu Removal Reaction of Compound 15a (TMSOTf-HMDS Method)

30.0 mg of Substrate 15a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (3.6 eq) and TMSOTf (2.4 eq)were sequentially added to the solution at room temperature. Five hoursafter the addition of the reagents, HMDS (1.8 eq.) and TMSOTf (1.2 eq.)were further added. One hour after the further addition of the reagents,the reaction was analyzed by LCMS and the raw material was confirmed todisappear. At this time, amide bond cleavage was not confirmed.

Example 51

t-Bu Removal Reaction of Compound 16a (TFA Method)

30.2 mg of Substrate 16a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA(20 eq.) at room temperature. 4.5 hours after the addition of thereagent, TFA (10 eq.) was further added. One hour after the furtheraddition of the reagent, the reaction was analyzed by LCMS to find thatthe reaction conversion rate was 65%. 7.0% of a by-product due to amidebond cleavage was observed.

Example 52

t-Bu Removal Reaction of Compound 16a (TMSOTf-HMDS Method)

29.9 mg of Substrate 16a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (3.6 eq) and TMSOTf (2.4 eq)were sequentially added to the solution at room temperature. Four hoursafter the addition of the reagents, the reaction was analyzed by LCMSand the raw material was confirmed to disappear. At this time, amidebond cleavage was not confirmed.

Example 53

t-Bu Removal Reaction of Compound 17a (TFA Method)

50.0 mg of Substrate 17a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA (5eq.) at 0° C. Two hours after the addition of the reagent, TFA (5 eq.)was added. After another 15.5 hours, the reaction solution was warmed toroom temperature. Three hours after warming, TFA (10 eq.) was added.After another 1.5 hours, TFA (20 eq.) was added. 1.5 hours after thefurther addition of the reagent, the reaction was analyzed by LCMS tofind that the reaction conversion rate was 54%. 26% of a by-product dueto amide bond cleavage was observed.

Example 54

t-Bu Removal Reaction of Compound 17a (TMSOTf-HMDS Method)

30.0 mg of Substrate 17a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (5.4 eq.) and TMSOTf (3.6 eq)were sequentially added to the solution at 0° C. Four hours after theaddition of the reagents, the reaction solution was warmed to roomtemperature. Three hours after warming, the reaction was analyzed byLCMS and the raw material was confirmed to disappear. At this time,amide bond cleavage was not confirmed.

Example 55

t-Bu Removal Reaction of Compound 18a (TFA Method)

29.9 mg of Substrate 18a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA(20 eq.) at 0° C. 4.5 hours after the addition of the reagent, TFA (10eq.) was further added and the reaction solution was warmed to roomtemperature. Two hours after the further addition of the reagent, thereaction was analyzed by LCMS to find that the reaction conversion ratewas 82%. 34% of a by-product due to amide bond cleavage was observed.

Example 56

t-Bu Removal Reaction of Compound 18a (TMSOTf-HMDS Method)

29.8 mg of Substrate 18a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (7.2 eq.) and TMSOTf (4.8 eq)were sequentially added to the solution at 0° C. Three hours after theaddition of the reagents, the reaction solution was warmed to roomtemperature. 3.5 hours after warming, the reaction was analyzed by LCMSand the raw material was confirmed to disappear. At this time, amidebond cleavage was not confirmed.

Example 57

t-Bu Removal Reaction of Compound 19a (TFA Method)

50.0 mg of Substrate 19a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA (5eq.) at 0° C. Two hours after the addition of the reagent, TFA (5 eq.)was added. After another 15.5 hours, the reaction solution was warmed toroom temperature. Three hours after warming, TFA (10 eq.) was added.After another 1.5 hours, TFA (20 eq.) was added. 1.5 hours after thefurther addition of the reagent, the reaction was analyzed by LCMS tofind that the reaction conversion rate was 82%. 14% of a by-product dueto amide bond cleavage was observed.

Example 58

t-Bu Removal Reaction of Compound 19a (TMSOTf-HMDS Method)

30.0 mg of Substrate 19a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (3.6 eq.) and TMSOTf (2.4 eq)were sequentially added to the solution at 0° C. 3.5 hours after theaddition of the reagents, the reaction solution was warmed to roomtemperature and HMDS (3.6 eq.) and TMSOTf (2.4 eq.) were further added.One hour after the further addition of the reagents, the reaction wasanalyzed by LCMS and the raw material was confirmed to disappear. Atthis time, amide bond cleavage was not confirmed.

As shown in Examples 45 to 58, it was found that in tBu removalreactions, the TMSOTf-HMDS method using a combination of silylatingagents and electrophilic species scavengers does not cause main chaindamage and allows deprotection reactions to proceed more efficiently, ascompared with the conventional TFA method.

TABLE 113 Reaction conditions and experimental results ExampleConversion Purity reduction Main chain cleavage Substrate No. Reactioncondition rate (LCMS Area %) (LCMS Area %) 13a 45 TFA (10 to 20 eq.) 52%22% MePhe-MeVal: 10.0% CH₂Cl₂ (10 v/w) rt, 6 h 46 TMSOTf (2.4 to 4.8eq.) >99%  1.2%  Not detected. HMDS (3.6 to 7.2 eq.) EtOAc (10 v/w %) 0°C. to rt, 5 h 14a 47 TFA (20 eq.) 92% 46% MePhe-MeVal: 22.0% CH₂Cl₂ (10v/w) MeVal-Asp(pip): 1.5% rt, 7 h 48 TMSOTf (2 eq.) 100%  0.7%  Notdetected. HMDS (3 eq.) EtOAc (10 v/w %) 0° C., 6 h 15a 49 TFA (20 eq.)50% 33% MeIle-Ser(tBu): 6.4% CH₂Cl₂ (10 v/w) MePhe-MeVal: 16.3% rt, 6 hMeVal-Asp(pip): 5. 0% 50 TMSOTf (2.4 to 3.6 eq.) 100%  0.2%  Notdetected. HMDS (3.6 to 5.4 eq.) EtOAc (10 v/w %) rt, 6 h 16a 51 TFA (20to 30 eq.) 65% 26% MeIle-tBuSer: 7.0% CH₂Cl₂ (10 v/w) rt, 6 h 52 TMSOTf(2.4 eq.) 100%  ≈0% Not detected. HMDS (3.6 eq.) EtOAc (10 v/w %) rt, 4h 17a 53 TFA (5 to 40 eq.) 54% 25% MeIle-Ser(tBu): 5.8% CH₂Cl₂ (10 v/w)MeVal-Asp(pip): 20.5% 0° C. to rt, 24 h 54 TMSOTf (3.6 eq.) 100%  2.6% Not detected. HMDS (5.4 eq.) EtOAc (10 v/w %) 0° C. to rt, 7 h 18a 55TFA (20 to 30 eq.) 82% 33% MePhe-MeVal: 15.1% CH₂Cl₂ (10 v/w)MeVal-Asp(pip): 12.3% 0° C. to rt, 7 h MeLeu-Val: 6.9% 56 TMSOTf (4.8eq.) 100%  2.7%  Not detected. HMDS (7.2 eq.) EtOAc (10 v/w %) 0° C. tort, 7 h 19a 57 TFA (5 to 40 eq.) 54% 17% MeIle-Ser(tBu): 0.8% CH₂Cl₂ (10v/w) MePhe-Leu: 5.4% 0° C. to rt, 24 h MePhe-MeVal: 6. 6% MeLeu-Val:1.6% 58 TMSOTf (2.4 to 4.8 eq.) 100%  0.6%  Not detected. HMDS (3.6 to7.2 eq.) EtOAc (10 v/w %) 0° C. to rt, 5 h

Identification of Products

TABLE 114 (HPLC method 1) MS (major peaks) and retention times of thetarget products (in Examples of the TMSOTf/HMDS method) Compound dataRetention Example Compound time No. No. MW m/z (min) 45, 46 13b 751.92752.42 ([M + H]+) 4.723 47, 48 14b 879.11 879.52 ([M + H]+) 5.313 49, 5015b 1049.32 1049.63 ([M + H]+) 4.985 51, 52 16b 1176.51 1198.67 ([M +Na]+) 5.527 53, 54 17b 1275.64 1089.62 5.652 55, 56 18b 1450.87 536.235.964 57, 58 19b 1535.98 1222.41 6.183

TABLE 115 MS and retention times of decomposed products Example Compounddata No. Compound No. MW m/z Retention time (min) 45 13c + its isomer456.54 457.28 ([M + H]+) 4.504 439.30 ([M—H20 + H]+) 4.894 13d + itsisomer 369.51 370.29 ([M + H]+) 2.691 370.31 ([M + H]+) 2.731 47 14c696.89 679.35 ([M—H20 + H]+) 4.935 14d + its isomer 583.73 584.20 ([M +H]+) 5.118 566.19 ([M—H20 + H]+) 5.477 49 15c + its isomer 449.55 432.28([M—H20 + H]+) 5.287 450.30 ([M + H]+) 3.928 15d 673.90 674.40 ([M +H]+) 3.792 15e 753.94 754.29 ([M + H]+) 4.863 15f + its isomer 697.83698.35 ([M + H]+) 4.101 736.93 736.30 ([M—H20 + H]+) 5.123 15g + itsisomer 810.99 793.37 ([M—H20 + H]+) 4.487 793.40 ([M—H20 + H]+) 5.444 5116c 673.90 674.48 ([M + H]+) 3.591 16d + its isomer 576.74 577.32 ([M +H]+) 4.500 559.28 ([M—H20 + H]+) 5.267 53 17c + its isomers 689.90690.45 ([M + H]+) 4.764 672.38 ([M—H20 + H]+) 5.432 672.59 ([M—H20 +H]+) 5.463 17d + its isomer 1107.45 1129.61 ([M + Na]+) 5.627 17e + itsisomers 1051.34 1033.66 ([M—H20 + H]+) 5.116 1033.77 ([M—H20 + H]+)5.944 1033.64 ([M—H20 + H]+) 5.985 55 18c 553.70 536.31 ([M—H20 + H]+)5.199 18d 1212.54 1194.58 ([M—H20 + H]+) 5.469 18e + its isomers 1155.491177.57 ([M + Na]+) 5.746 1177.39 ([M + Na]+) 5.788 1137.73 ([M—H20 +H]+) 5.904 57 19c 673.90 674.38 ([M + H]+) 3.633 19d + its isomer1155.53 1155.73 ([M + H]+) 3.863, 3.906 19e 1211.64 1211.65 ([M + H]+)4.619 19f 398.46 381.16 ([M—H20 + H]+) 4.898 19g 638.81 621.31 ([M—H20 +H]+) 5.246 19h + its isomers 1240.60 1240.51 ([M + H]+) 5.794, 5.8311222.59 ([M—H20 + H]+) 5.953

The structures of Compounds 13c to 19h are provided below.

Example 59

t-Bu Removal Reaction of Fmoc-MeAsp(OtBu)-pip (Compound 12a) (1-mer: TFAMethod)

48.0 mg of the raw material was weighed into a reaction vessel anddissolved in 9 v/w of dichloromethane. 1 v/w of trifluoroacetic acid wasthen added and the reaction solution was stirred. 19 hours after theaddition of the reagents, the reaction was analyzed by LCMS (HPLC method4) and the conversion rate (=target product/(target product+startingmaterial)) was confirmed to be 77%. At this time, a rearrangementproduct 12c and a hydrolysate 12d were observed as compounds in whichamide bonds were damaged. Impurities of unknown structure were alsoproduced.

TABLE 116 Weight (mg) Yield (%) Raw material (Compound 12a) 48.0 —Product (Compound 12b) Not isolated Not isolated

Raw Material Purity Analysis (HPLC Method 1)

TABLE 117 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material(Compound 12a) 492.26 493.24 ([M + H]+) 5.527 98.268

TABLE 118 Analysis (HPLC method 4) MW m/z rt Purity LC A % Raw material(Compound 12a) 492.26 437.25 ([M-tBu + H]+) 23.660 98.268 Product(Compound 12b) 436.20 437.22 ([M + H]+) 15.194 53.776 Rearrangementproduct (Compound 12c) 436.20 437.28 ([M + H]+) 15.553 1.474 Hydrolysate(Compound 12d) 369.12 250.91 10.522 0.901 Impurity of unknown structure(12e) 102.07 17.949 28.043

Example 60

t-Bu removal reaction of Fmoc-MeAsp(OtBu)-pip (Compound 12a) (1-mer:TMSOTf-HMDS Method)

The experiment was performed according to Example 2.

TABLE 119 Weight (g) Yield (%) Raw material (Compound 12a) 59.9 —Product (Compound 12b) 55.8 Quantitative

TABLE 120 Analysis (HPLC method 1) MW m/z rt Purity LC A % Raw material(Compound 12a) 492.26 493.24 ([M + H]+) 5.527 98.268

TABLE 121 Analysis (HPLC method 4) MW m/z rt Purity LC A % Product(Compound 12b) 436.20 437.22 ([M + H]+) 15.194 98.999 Rearrangementproduct (Compound 12c) 436.20 437.28 ([M + H]+) 15.553 0.379 Hydrolysate(Compound 12d) 369.12 250.91 10.522 0.15 Impurity of unknown structure(12e) 102.07 17.949 0.474E. Experiment of Comparison Between the TFA Method and the TMSOTf-HMDSMethod in t-Bu Removal Reactions (Table 122)

Example 61

t-Bu Removal Reaction of Compound 20a (TFA Method)

27.1 mg of Substrate 20a was weighed into a reaction vessel anddissolved in 10 v/w of dichloromethane. To the solution was added TFA(10 eq.) at room temperature. Eight hours after the addition of thereagent, the reaction was analyzed by LCMS to find that the reactionconversion rate was 76%. The purity was reduced by 11%, while 8.1% of aby-product due to amide bond cleavage was confirmed. The same reactionwas performed for Substrate 21a (Table 122).

Example 62

t-Bu Removal Reaction of Compound 20a (TMSOTf-HMDS Method)

27.6 mg of Substrate 20a was weighed into a reaction vessel anddissolved in 10 v/w of ethyl acetate. HMDS (2.4 eq) and TMSOTf (2.4 eq)were sequentially added to the solution at room temperature. Two hoursafter the addition of the reagents, the reaction was analyzed by LCMSand the raw material was confirmed to disappear. At this time, amidebond cleavage was not confirmed.

The same reaction was performed for Substrate 21a (Table 122).

TABLE 122 Purity Amide bond Example Raw Reaction Conversion reductioncleavage Substrate No. material time rate (LCMS Area %) (LCMS Area %)20a 61 27.1 mg 8 h  76% 11%  8.1% 62 27.6 mg 2 h 100% 0% Not detected.21a 63 26.0 mg 8 h  76% 9% 2.7% 64 26.2 mg 2 h 100% 0% Not detected.

Identification of Products

TABLE 123 (HPLC method 1) Major peaks and retention times of productsand target products Example Compound data No. Compound MW m/z Retentiontime (min) 61 20b 410.47 433.16 ([M + Na]+)    3.858 20c 325.36 104.01([M-Fmoc + H]+)*  3.976 63 21b 486.57 487.17 ([M + Na]+)    4.485 21c401.46 180.02 ([M-Fmoc + H]+)** 4.594

F. Experiment of Comparison Between the TFA Method and the TMSOTf-HMDSMethod in Resin Removal Reactions (Table 124)

Example 65

Resin Removal Reaction of Resin 22a (TFA Method)

30.2 mg of Resin 22a was weighed into a reaction vessel, and the resinwas swollen by adding 10 v/w of dichloromethane. After removing thedichloromethane from the reaction solution, 10 v/w % of TFA (5%dichloromethane solution) was added at room temperature and the vesselwas shaken at 25° C. One hour after the shaking started, the reactionwas analyzed by LCMS to find that 7.8% of a by-product due to amide bondcleavage was produced. The same reaction was performed for Resins 23a to24a (Table 124).

Example 66

Resin Removal Reaction of Resin 22a (TMSOTf-HMDS Method)

23.0 mg of Substrate 22a was weighed into a reaction vessel, and theresin was swollen by adding 10 v/w of 1,2-dichloroethane. After removingthe 1,2-dichloroethane from the reaction solution, a solution of HMDS(3.6 eq.) and TMSOTf (2.4 eq.) in 10 v/w of 1,2-dichloroethane was addedto the reaction vessel. After stirring at 25° C. for four hours, thereaction was analyzed by LCMS and no amide bond cleavage was confirmed.The same reaction was performed for Resins 23a to 24a (Table 124).

TABLE 124 Amide bond Example Raw Reaction cleavage Substrate No.material Reagent amount time (LCMS Area %) 22a 65 30.2 mg 5% TFA in 1 h7.9% CH₂Cl₂ (10 v/w) 66 23.0 mg TMSOTf (2.4 eq.) 4 h  0% HMDS (4.8 eq.)23a 67 30.0 mg 5% TFA in 1 h 2.3% CH₂Cl₂ (10 v/w) 68 24.0 mg TMSOTf (2.4eq.) 4 h  0% HMDS (4.8 eq.) 24a 69 30.3 mg 5% TFA in 1 h 0.3% CH₂Cl₂ (10v/w) 70 23.6 mg TMSOTf (2.4 eq.) 4 h  0% HMDS (4.8 eq.)

Identification of Products

HPLC Method A

-   -   Instrument: Waters ACQUITY UPLC H-Class    -   Column: Ascentis Express C18 (2.7 μm, 4.6 mm×50 mm), Supelco    -   Eluent: A) 0.05% TFA/water, B) 0.05% TFA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (4 min.)⇒100% (4.5 min.)⇒5% (4.6        min.) ⇒5% (6 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm    -   Injection vol.: 5 μL    -   Sample prep.: 25 μL/0.975 mL MeCN

HPLC Method B

-   -   Instrument: Waters ACQUITY UPLC H-Class    -   Column: Ascentis Express C18 (2.7 μm, 4.6 mm×50 mm), Supelco    -   Eluent: A) 0.05% TFA/water, B) 0.05% TFA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (4 min.)⇒100% (4.5 min.)⇒5% (4.6        min.)⇒    -   5% (6 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm    -   Injection vol.: 5 μL    -   Sample prep.: 5 μL/1.00 mL (MeCN 0.950 ml+0.1 M phosphate buffer        (pH 8.0) 0.050 ml)

HPLC Method C

-   -   Instrument: Waters ACQUITY UPLC H-Class    -   Column: Ascentis Express C18 (2.7 μm, 4.6 mm×50 mm), Supelco    -   Eluent: A) 0.05% TFA/water, B) 0.05% TFA/CH3CN Gradient (B): 5%        (0 min.)⇒100% (4 min.)⇒100% (4.5 min.)⇒5% (4.6 min.)⇒5% (6 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm    -   Injection vol.: 5 μL    -   Sample prep.: 5 μL/1.00 mL MeCN

HPLC Method D

-   -   Instrument: Shimadzu LCMS-2020    -   Column: Ascentis Express C18 (2.7 μm, 2.1 mm×50 mm), Supelco    -   Eluent: A) 0.1% FA/water, B) 0.1% FA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (4.5 min.)⇒100% (5 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm-400 nm    -   Injection vol.: 1 μL

HPLC Method E

-   -   Instrument: SHIMADZU LCMS-2020    -   Column: Ascentis Express C18 2.1 mm×50 mm, Supelco    -   Eluent: A) 0.1% FA/water, B) 0.1% FA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (1.5 min.)⇒100% (2 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: PDA 210 nm-400 nm    -   Injection vol.: 1 μL

HPLC Method F

-   -   Instrument: Waters Acquity UPLC/SQD2    -   Column: Ascentis Express C18 (2.7 μm, 2.1 mm×50 mm), Supelco    -   Eluent: A) 0.1% FA/water, B) 0.1% FA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (1 min.)⇒100% (1.4 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm-400 nm    -   Injection vol.: 1 μL

HPLC Method G

-   -   Instrument: Waters Acquity UPLC/SQD2    -   Column: Ascentis Express C18 (2.7 μm, 2.1 mm×50 mm), Supelco    -   Eluent: A) 0.1% FA/water, B) 0.1% FA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (1 min.)⇒100% (1.4 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm-400 nm    -   Injection vol.: 1 μL

HPLC Method H

-   -   Instrument: Waters Acquity UPLC/SQD    -   Column: Ascentis Express C18 (2.7 μm, 2.1 mm×50 mm), Supelco    -   Eluent: A) 0.1% FA/water, B) 0.1% FA/CH3CN    -   Gradient (B): 5% (0 min.)⇒100% (4.5 min.)⇒100% (5.0 min.)    -   Flow rate: 1.0 mL/min.    -   Detection: 210 nm-400 nm    -   Injection vol.: 2 μL

TABLE 125 Example HPLC Compound data No. method Compound MW m/zRetention time (min) 65

A 20b 410.47 411.18 ([M + H]+)    3.034 66 A 22c 339.39 118.01([M-Fmoc + H]+) 3.648 67

A 23b 438.52 439.24 ([M + H]+)    3.310 68 A 23c 353.42 354.16 ([M +H]+)    3.472 69

B 24b 422.48 423.19 ([M + H]+)    2.898 70 B 24c 337.38 116.01([M-Fmoc + H]+) 3.049

G. Experiment of Comparison Between the TFA Method and the TMSOTf-HMDSMethod in Resin Removal Reactions (Table 126)

Example 71

CTC Resin Removal and t-Bu Removal Reactions of Resin 25a (TFA Method)

20.0 mg of Resin 25a was weighed into a reaction vessel, and the resinwas swollen by adding 10 v/w of dichloromethane. After removing thedichloromethane from the reaction solution, 10 v/w % of TFA (10%dichloromethane solution) was added at room temperature and the vesselwas shaken at 25° C. One hour after the shaking started, the reactionwas analyzed by LCMS to find that the reaction conversion rate was 17%.3.8% of a by-product due to amide bond cleavage was produced. The samereaction was performed for Resin 26a (Table 126).

Example 72

CTC Resin Removal and tBu Deprotection Reactions of Resin 25a(TMSOTf-HMDS Method)

23.0 mg of Substrate 25a was weighed into a reaction vessel, and theresin was swollen by adding 10 v/w of 1,2-dichloroethane. After removingthe 1,2-dichloroethane from the reaction solution, a solution of HMDS(10.7 eq.) and TMSOTf (7.1 eq.) in 10 v/w of 1,2-dichloroethane wasadded to the reaction vessel. After shaking at 25° C. for four hours,the reaction was analyzed by LCMS and the t-Bu ester was confirmed todisappear. The same reaction was performed for Resin 26a (Table 126).

TABLE 126 Amide bond Example Raw Reaction Conversion cleavage SubstrateNo. material Reagent time rate (LCMS Area %) 25a 71 20.0 mg 10% TFA 1 h 17% 3.8% in CH₂Cl₂ (10 v/w) 72 20.3 mg TMSOTf (7.1 eq.) 4 h 100%   0%HMDS (10.7 eq.) 26a 73 19.8 mg 10% TFA 1 h  13% 1.1% in CH₂Cl₂ (10 v/w)74 20.0 mg TMSOTf (7.1 eq.) 3 h 100%   0% HMDS (14.2 eq.)

Identification of Products

TABLE 127 (HPLC method 1) Major peaks and retention times of productsand target products Compound data Example No. Compound MW m/z Retentiontime (min) 71, 72 25b 454.48 477.10 ([M + Na]+)   2.649 25c 339.39118.07 ([M-Fmoc + H]+) 3.560 73, 74 26b 496.56 497.24 ([M + H]+)   3.117 26c 367.45 146.00 ([M-Fmoc + H]+) 3.661

H. Synthesis of Raw Materials for Boc Removal, t-Bu Removal, and ResinRemoval Reactions

Example 75

Synthesis of 3-Mer Raw Material for Boc Removal Reaction(Boc-Asp(OBn)-pip)

The synthesis was performed according to Example 88.

TABLE 128 Weight (g) Yield (%) Raw material (27) 34.6 — Product (28)37.6 90

TABLE 129 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (28)390.48 291.19 ([M-Boc + H]+) 4.530 97.619

Example 76

Synthesis of 2-mer (Boc-MeVal-Asp(OBn)-pip)

5.714 g of the raw material was weighed into a reaction vessel and 29 mlof MeCN was added. After adding 2.3 ml of methanesulfonic acid, the oilbath was set at 45° C. and the reaction vessel was heated. After 70minutes, the reaction vessel was brought back to room temperature andthen immersed in an ice bath. To the reaction solution was added 14 mlof diisopropylethylamine, followed by addition of 4.046 g ofBoc-MeVal-OH and 5.284 g of DMT-MM. One hour after the addition of thereagents, the reaction solution was concentrated. The reaction wasquenched by adding ethyl acetate and 5% aqueous potassium carbonate tothe concentrate. The organic layer was separated by liquid separationtreatment and then washed twice with a 5% aqueous potassium carbonatesolution, once with water, three times with a 5% aqueous potassiumbisulfate solution, once with a 10% aqueous sodium chloride solution,and once with brine. The organic layer was concentrated under reducedpressure to give 7.055 g (96% yield) of a pale yellow solid.

TABLE 130 Weight (g) Yield (%) Raw material (28) 5.714 — Product (29)7.055 96

TABLE 131 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (29)503.64 526.29 ([M + Na]+) 5.178 96.889

Example 77

Synthesis of 3-mer (Boc-MePhe-MeVal-Asp(OBn)-pip)

7.055 g of the raw material was weighed into a reaction vessel and 35 mlof MeCN was added. After adding 2.2 ml of methanesulfonic acid, the oilbath was set at 45° C. and the reaction vessel was heated. After 70minutes, the reaction vessel was brought back to room temperature andthen the reaction solution was concentrated. After the concentration, 35ml of ethyl acetate was added, the reaction vessel was immersed in anice bath, and 9.466 g of sodium bicarbonate was then added. 35 ml ofwater was then added, followed by addition of 4.61 g of Boc-MePhe-OH and7.73 g of DMT-MM. After six hours, the reaction was followed by LCMS toconfirm that the raw material consumption rate was 99%. One hour afterthe analysis, liquid separation treatment was conducted by adding ethylacetate and water to give an organic layer. The organic layer was washedthree times with a 5% aqueous potassium carbonate solution, once withwater, three times with a 5% aqueous potassium bisulfate solution, andonce with brine. The organic layer was concentrated under reducedpressure and the resulting crude product was purified by silica gelcolumn chromatography to provide 6.9757 g (75% yield) of the targetproduct as a white solid.

TABLE 132 Weight (g) Yield (%) Raw material (29) 7.055 — Product (9a)6.9757 75

TABLE 133 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (9a)664.84 687.43 ([M + Na]+) 5.684 99.206

Example 78

Synthesis of Boc-MeGly-OAllyl

9.286 g of the raw material was weighed into a reaction vessel and 46 mlof DMF was added. The reaction vessel was cooled with ice, and 10.20 gof potassium carbonate and 4.15 ml of allyl bromide were then added.After 15 minutes, the reaction vessel was brought back to roomtemperature and then stirred overnight. To the reaction vessel was addedMTBE, and the reaction was quenched with water. After liquid separation,the organic layer was washed once with brine. The organic layer wasdried over magnesium sulfate, filtered, and concentrated under reducedpressure. The resulting crude product was purified by silica gel columnchromatography to provide 10.083 g (90% yield) of the target product 31as a transparent oily liquid.

TABLE 134 Weight (g) Yield (%) Raw material (30) 9.286 — Product (31)10.083 90

Example 79

Synthesis of Boc-Thr(OBn)-MeGly-OAllyl

Deprotection and condensation reactions were performed according toExample 76.

TABLE 135 Weight (g) Yield (%) Raw material (31) 6.5224 — Product (32)13.5594 Quantitative

TABLE 136 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product420.51 321.10 ([M-Boc + H]+) 4.770 98.54 (Compound 32)

Example 80

Synthesis of Boc-MeLeu-Thr(OBn)-MeGly-OAllyl

Deprotection and condensation reactions were performed by the samemethod as described above.

Example 80a

Synthesis of Teoc-MeLeu-OPfp by Esterification Reaction of Teoc-MeLeu-OHwith Pentafluorophenol (Pfp-OH)

926 mg of the raw material and 736 mg of pentafluorophenol (Pfp-OH) wererespectively weighed into a reaction vessel, and 7.8 mL of isopropylacetate was added. 767 mg of EDCI hydrochloride was added and thereaction solution was stirred at room temperature for two hours. Theorganic layer was washed twice with 8 mL of 0.5 N hydrochloric acid andtwice with 8 mL of a 5% aqueous potassium carbonate solution, and 2 g ofsodium sulfate was added to the organic layer. The solid was removed byfiltration and the organic layer was concentrated under reduced pressureto give the target product as a transparent oily liquid. The obtainedcompound is used for the next reaction without purification.

TABLE 137 Analysis (HPLC method G) Retention time MW m/z (min) Product455.16 428.3 ([M—C2H4 + H]+) 1.19 (Compound 80a)

Example 80b

Synthesis of Teoc-MeLeu-Thr(OBn)-MeGly-OAllyl

Compound 80b can be synthesized by condensation reaction using themethod of Meneses et al. (J. Org. Chem., 2010, 75, 564-569) in which theterminal protecting group of Compound 33 is removed and then reactedwith pentafluorophenyl ester (Compound 80a) to form an amide bond.

TABLE 138 Weight (g) Yield (%) Raw material (33) 13.4338 — Product (8a)15.5319 100

TABLE 139 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (8a)660.41 561.28 ([M-Boc + H]+) 5.657 95.36

Example 81

Synthesis of Boc-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OAllyl

The reaction was performed according to Example 88.

TABLE 140 Weight (g) Yield (%) Raw material (4b) 5.0118 — Product (3a)5.68 90

TABLE 141 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (3a)906.55 807.58 ([M-Boc] + H)+ 5.974 93.06

Example 82

Synthesis of Boc-MeAla-MePhe-Leu-MeLeu-Thr(OBn)-MeGly-OH

1.1180 g of the raw material was weighed into a reaction vessel and 2.2ml each of THF, methanol, and water were added. 102.8 mg of lithiumhydroxide monohydrate was then added and the reaction solution wasstirred at room temperature for five hours. The completion of thereaction was confirmed by LC, and the reaction solution was thenconcentrated under reduced pressure. After adding ethyl acetate to theconcentrate, 0.5 N hydrochloric acid was added to quench the reaction.The organic layer was separated by liquid separation treatment and thenwashed twice with 0.5 N hydrochloric acid and twice with a 5% aqueoussodium chloride solution. The solvent was evaporated by concentrationunder reduced pressure to give 918.8 mg (86% yield) of a white solid.

TABLE 142 Weight (g) Yield (%) Raw material (3a) 1.1180 — Product (34)0.9188 86

TABLE 143 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (34)866.52 767.5 ([M-Boc] + H)+ 5.361 92.03

Example 83

Synthesis of Fmoc-Asp(OtBu)-pip

The reaction was performed according to Example 88.

TABLE 144 Weight (g) Yield (%) Raw material (11) 40 — Product (12) 47Quantitative

TABLE 145 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (12)478.25 423.24 ([M-tBu] + H)+ 4.019 99.318

Example 84

Synthesis of FmocAsp(OH)pip

45.4 g of the raw material was weighed into a reaction vessel and 454 mlof trifluoroethanol was added. 24 ml of chlorotrimethylsilane was thenadded and the reaction solution was stirred at room temperature. Two anda half hours after the start of the reaction, 910 ml of water was addedin two portions and crystals were allowed to precipitate. The crystalswere filtered and then dried to give 25.8 g (61% yield) of the targetproduct 36.

TABLE 146 Weight (g) Yield (%) Raw material (35) 40 — Product (36) 25.861

TABLE 147 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (36)422.18 423.21 ([M] + H)+ 3.101 98.259

Example 85

Loading of Fmoc-Asp(OH)-pip onto CTC Resin

The same loading step as in the method described in Example 112 wasconducted. The product was analyzed after 5-mer elongation and thefollowing resin removal step.

TABLE 148 Weight (g) Raw material (36) 4.09

Example 86

Solid-Phase Synthesis (2-mer Elongation)

The reactions were performed in a solid-phase synthesis column.

-   -   1) Deprotection step: To Fmoc-MeAla-OCTC resin (37, theoretical        amino acid loading amount: 9.49 mmol) was added DMF (104 mL). A        20% solution of piperidine in DMF (104 mL) was added at 30° C.,        and the column was shaken at 30° C. for 15 minutes. After the        solution was discharged, a 20% solution of piperidine in DMF        (104 mL) was added again and the column was shaken at 30° C. for        15 minutes. After the solution was discharged, the resin was        washed with DMF (130 ml) for 2 min×7.    -   2) Elongation step: In a reaction vessel separate from the        solid-phase column, 6.62 g of Fmoc-MeVal-OH and 2.93 g of Oxyma        were dissolved in 52 ml of DMF, and 5.87 ml of DIC was then        added. The vessel was shaken at 30° C. for 30 minutes. This        solution was added to the solid-phase synthesis column, which        was then shaken at 30° C. for two hours. After the solution was        discharged, the resin was washed with DMF (130 ml) for 2 min×6.

Example 87

Solid-Phase Synthesis (Sequential Elongation from 2-mer to 5-mer andResin Removal Reaction)

Deprotection and elongation steps were repeated according to the methoddescribed in Example 86 to provideBoc-MeIle-Ala-MePhe-MeVal-Asp(OCTC)-pip. To the resulting resin wasadded 147 ml of a mixed solvent of diisopropylethylamine,trifluoroethanol, and methylene chloride (2% diisopropylethylamine,solvent volume ratio 1:1), followed by shaking at 30° C. for two and ahalf hours. The discharged and resin-removed peptide solution wascollected. After the resin removal solution was discharged, the resinwas washed twice with 25 ml of a mixed solvent of trifluoroethanol andmethylene chloride (volume ratio 1:1), and the solutions wereconcentrated. The concentrate was dissolved in 130 ml of ethyl acetateand washed twice with an aqueous potassium carbonate solution, twicewith potassium bisulfate, and once with an aqueous sodium chloridesolution, and the organic layer was then concentrated under reducedpressure. After drying, 4.9821 g (69% yield) of a white solid wasobtained.

TABLE 149 Weight (g) Yield (%) Fmoc-MeAsp(OH)-pip (37) 4.09 — Product(38) 4.9821 69

TABLE 150 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product (38)772.47 795.5 ([M + Na]+) 4.856 96.471

Example 88

Synthesis of Fmoc-MeAsp(OtBu)-pip

49.6 g of EDCI hydrochloride was weighed into a reaction vessel and 400ml of DMF was added. The reaction vessel was cooled to 0° C., and100.050 g of Fmoc-MeAsp(OtBu)-OH was then added. Next, a solution of40.1 g of Oxyma in 100 ml of DMF was added, followed by 34.9 ml ofpiperidine, using a dropping funnel. Four hours and 30 minutes after thedropwise addition of piperidine was completed, the reaction was analyzedby LCMS and the raw material was confirmed to disappear. The reactionsolution was diluted with ethyl acetate, and 500 ml of 0.5 Nhydrochloric acid was then added to quench the reaction. The organiclayer was separated and then washed twice with water, twice with a 5%aqueous sodium carbonate solution, and twice with a 5% aqueous sodiumchloride solution. The resulting organic layer was concentrated underreduced pressure. The resulting crude product was recrystallized fromethyl acetate and heptane to give 86.230 g (75% yield) of the targetproduct.

TABLE 151 Analysis (HPLC method 1) MW m/z rt Purity LC A % Product492.26 493.24 ([M + H]+) 5.527 98.268 (Compound 11)

Example 89

Synthesis of Synthetic Intermediate (Cbz-Asp(OtBu)-pip (41)) for11-Residue Peptide (tBu form,Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip(22))

17.5 g of Cbz-Asp(OtBu)-OH was weighed into a reaction vessel and 175 mLof ethyl acetate was added. After cooling the reaction solution to 0°C., piperidine (3 eq.), diisopropylethylamine (6 eq.), and a 1.7 Msolution of T3P in ethyl acetate (3 eq.) were added, respectively. Thereaction solution was warmed to room temperature and stirred at roomtemperature for 10 minutes, and 175 mL of a 5% aqueous potassiumcarbonate solution was then added. The aqueous layer was removed, andthe organic layer was then washed twice with 175 mL of a 5% aqueouspotassium bisulfate solution. The resulting organic layer wasconcentrated and dried under reduced pressure to afford 20 g ofCbz-Asp(OtBu)-pip (41) in 100% yield.

Example 90 Synthesis of H-Asp(OtBu)-pip (42)

9.5 g of Cbz-Asp(OtBu)-pip (41) was weighed into each of two reactionvessels, and 50 mL of CPME was added to each vessel. 10% Pd/C (20 w/w %)was added to both vessels and then reacted at 30° C. under a 3 barhydrogen atmosphere. Three hours after the start of the reaction, thetwo reaction solutions were mixed, filtered, and washed with 100 mL ofCPME. The resulting mixture was concentrated and dried under reducedpressure to afford 12.3 g of H-Asp(OtBu)-pip (42) in 99% yield.

11-mer (22) was synthesized from H-Asp(OtBu)-pip (42) by the 19 stepsdescribed below. The synthesized intermediates are provided as follows.

(42) H-Asp(OtBu)-pip (43) Cbz-MeVal-Asp(OtBu)-pip (44)H-MeVal-Asp(OtBu)-pip (45) Cbz-MePhe-MeVal-Asp(OtBu)-pip (46)H-MePhe-MeVal-Asp(OtBu)-pip (13) Cbz-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip(47) H-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip (14)Cbz-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip (48)H-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip (49)Cbz-MeGly-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip (50)H-MeGly-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip (15)Cbz-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal- Asp(OtBu)-pip (51)H-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)- pip (16)Cbz-MeLeu-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal- Asp(OtBu)-pip (52)H-MeLeu-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal- Asp(OtBu)-pip (17)Cbz-Leu-MeLeu-Val-MeGly-Melle-Ser(OtBu)-MePhe- MeVal-Asp(OtBu)-pip (53)H-Leu-MeLeu-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal- Asp(OtBu)-pip (18)Cbz-MePhe-Leu-MeLeu-Val-MeGly-Melle-Ser(OtBu)- MePhe-MeVal-Asp(OtBu)-pip(54) H-MePhe-Leu-MeLeu-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip (19) Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-Melle-Ser(OtBu)-MePhe-MeVal-Asp(OtBu)-pip

Example 91 Elongation of Peptide Chain (Elongation Method A)

8.6 g of 42 was weighed into a reaction vessel and 108 mL of CPME wasadded. Cbz-MeVal-OH (1.1 eq.) and diisopropylethylamine (3 eq.) wereadded, and a solution of BEP (1.5 eq.) in 21.5 mL of MeCN was thenadded. After stirring at room temperature for three minutes, 15 mL of a10% aqueous sodium bisulfate solution was added. The aqueous layer wasremoved, and 15 mL of a 5% aqueous potassium carbonate solution andtrimethylamine hydrochloride (3 eq.) were then added to the organiclayer at room temperature. The mixture was warmed to 40° C. and thenstirred at 40° C. for 90 minutes. After cooling to room temperature, theaqueous layer was removed and the resulting organic layer was thenwashed with 15 mL of a 5% aqueous potassium carbonate solution. Theresulting organic layer was concentrated and dried under reducedpressure to give 17 g of 43 quantitatively.

Example 92

Removal of N-Terminal Cbz (Deprotection Method A)

9.5 g of 43 was weighed into each of two reaction vessels, and 50 mL ofCPME was added to each vessel. 10% Pd/C (20 w/w %) was added to bothvessels and then reacted at 35° C. under a 3 bar hydrogen atmosphere.Two hours after the start of the reaction, the two reaction solutionswere mixed, filtered, and washed with 100 mL of CPME. The resultingmixture was concentrated and dried under reduced pressure to give 14 gof 44 in 100% yield.

Example 93

Elongation of Peptide Chain (Elongation Method B)

14 g of 44 was weighed into a reaction vessel, and 126 mL of CPME and 14mL of acetonitrile were added, respectively. Cbz-MePhe-OH (1.1 eq.),diisopropylethylamine (8 eq.), and a 1.7 M solution of T3P in ethylacetate (3 eq.) was sequentially added at room temperature. Afterstirring at room temperature for one hour, 140 mL of a 5% aqueouspotassium bisulfate solution was added. The aqueous layer was removed,and 140 mL of a 5% aqueous potassium carbonate solution andtrimethylamine hydrochloride (3 eq.) were then added at roomtemperature. After stirring at room temperature for 30 minutes, theaqueous layer was removed. The resulting organic layer was then washedwith 140 mL of a 5% aqueous potassium carbonate solution. The resultingorganic layer was concentrated and dried under reduced pressure to give24.1 g of 45 in 96% yield.

Example 94

Removal of N-Terminal Cbz (Deprotection Method B)

9.2 g of 15a was weighed into each of two reaction vessels, and 46 mL ofCPME was added to each vessel. 10% Pd/C (20 w/w %) was added to bothvessels and then reacted at 35° C. under a 5 bar hydrogen atmosphere.Six hours after the start of the reaction, the reaction vessels werecooled to room temperature and stored at room temperature overnightunder an air atmosphere. Further reaction was performed at 45° C. forfour hours under a 5 bar hydrogen atmosphere. The two reaction solutionswere mixed, filtered, and washed with 92 mL of CPME. The resultingmixture was concentrated and dried under reduced pressure to give 15.9 gof 51 in 98% yield.

The peptides of 2-mer to 11-mer were synthesized by the same technique.The reaction conditions different from those described above, except forthe reaction time and the mass of the raw material used, are furtherdescribed in the column “Reaction method.”

TABLE 152 (HPLC method 1) Example Raw Reaction Product Purity No.Product Reaction method material time amount Yield (LCMS Area %) 91 43Elongation A 8.6 g 3 min 17 g Quantitative 99.7% 92 44 Deprotection A9.5 g × 2 2 h 14 g 100%  ND 93 45 Elongation B 14 g 30 min 24.1 g 96%99.6% 95 46 Deprotection A 11.5 g × 2 2 h 18.1 g 99% ND 96 13 ElongationB 17.3 g 15 min 26.5 g Quantitative 98.9% 97 47 Deprotection A 12 g × 24 h 19.5 g 97% ND 98 14 Elongation A 16 g 5 min 22.2 g 100%  99.4% 99 48Deprotection A 9.5 g × 2 2 h 15.6 g 96% ND 100 49 Elongation B 15.3 g 15min 19.5 g Quantitative 99.6% 101 50 Deprotection A 9.5 g × 2 3 h 16.3 g99% ND (5 bar) 102 15 Elongation B 16 g 30 min 20 g 99% 99.6% 94 51Deprotection B 9.2 g × 2 10 h 15.9 g 98% ND 103 16 Elongation A 14.5 g 1min 18.0 g 98% 96.0% (40° C.) 104 52 Deprotection A 8 g × 2 4 h 14.3 g100%  ND (45° C., 5 bar) 105 17 Elongation B 13 g 30 min 15.6 g 98%97.2% 106 53 Deprotection A 10 g 4 h 8.9 g 99% ND (45° C., 5 bar) 107 18Elongation A 7 g 3 min 8.6 g 99% 97.0% 108 54 Deprotection A 7.6 g 4 h6.8 g 98% ND (45° C., 5 bar) 109 19 Elongation B 500 mg 2 h 555 mg 96%95.3%

Among the synthesized intermediates, 13a, 14a, 15a, 16a, 17a, 18a, and19a were used for tBu removal experiments. The compounds used for tBuremoval reactions were analyzed by LCMS as described in Table 153.

TABLE 153 Analysis Compound data Example HPLC Retention time No.Compound Method MW m/z (min) 46 13a 1 808.03 808.49 ([M + H]+) 5.938 4814a 1 935.22 935.59 ([M + H]+) 6.233 50 15a 1 1105.43 1105.69 ([M + H]+)5.924 52 16a 1 1232.62 1232.79 ([M + H]+) 6.374 54 17a 1 1345.78 1369([M + Na]+) 6.727 56 18a 1 1506.98 1530 ([M + Na]+) 7.096 58 19a 11592.09 1615 ([M + Na]+) 6.987I. Synthesis of C-Terminal tBu-Protected Dipeptides

Example 110

Synthesis of Fmoc-MeAla-MeAla-OtBu (20a)

99.7 mg of H-MeAla-OtBu hydrochloride and 710 mg of dipotassiumhydrogenphosphate were weighed in a reaction vessel, respectively, and10 v/w each of 2-MeTHF and water relative to H-MeAla-OtBu hydrochloridewere added. Fmoc-MeAla-OH (167 mg) and DMT-MM (211 mg) were sequentiallyadded at 0° C. and the reaction solution was then warmed to roomtemperature. After shaking at room temperature for 16 hours, thereaction was analyzed by LCMS and Fmoc-MeAla-OH was confirmed todisappear. The reaction solution was transferred to a separatory funnelusing 4 mL of water and 6 mL of 2-MeTHF, and the aqueous layer was thenremoved. The resulting organic layer was washed with brine (3 mL), 15%aqueous sodium bisulfate (3 mL), and 5% aqueous sodium carbonate (3 mL),respectively, and the solvent was then removed by concentration underreduced pressure. The resulting crude product was purified by silica gelcolumn chromatography using the conditions described below, and thefractions containing the target product were then concentrated and driedunder reduced pressure to provide 227.6 mg of 20a in a yield of 96% anda purity of 99.5%.

Example 111

Synthesis of Fmoc-MePhe-MeAla-OtBu(21a)

H-MeAla-OtBu hydrochloride (99.8 mg) and Fmoc-MePhe-OH (205 mg) wereweighed into a reaction vessel, respectively, and 8 v/w of isopropylacetate and 2 v/w of acetonitrile relative to H-MeAla-OtBu hydrochlorideas well as diisopropylethylamine (4 eq.) were sequentially added. A 1.7M solution of T3P in ethyl acetate (2.5 eq.) was added at roomtemperature. After stirring at room temperature for four hours, a 1.7 Msolution of T3P in ethyl acetate (0.5 eq.) and diisopropylethylamine(0.8 eq.) were added. 30 minutes after the addition of the reagents, thereaction was analyzed by LCMS to find that the reaction conversion ratewas 98.5%. NMI (2 eq.) was added at room temperature, and the reactionsolution was then stirred at 50° C. for five minutes. The reactionsolution was transferred to a separatory funnel using 6 mL of ethylacetate, and the aqueous layer was then removed. The resulting organiclayer was washed with a 5% aqueous sodium carbonate solution (3 mL), a5% aqueous potassium bisulfate solution (3 mL), and a 5% aqueous sodiumcarbonate solution (3 mL), respectively. The organic layer wasdehydrated with sodium sulfate for 30 minutes, then filtered, andconcentrated under reduced pressure to remove the solvent. The resultingcrude product was purified by silica gel column chromatography using theconditions described below, and the fractions containing the targetproduct were then concentrated and dried under reduced pressure toprovide 227.6 mg of 21a in a yield of 96% and a purity of 99.5%.

TABLE 154 Analysis (HPLC method 1) Compound data Example Retention timeNo. Compound MW m/z (min) 110 20a 466.58 489.2 ([M + Na]+) 5.243 111 21a542.68 543.2 ([M + H]+) 5.754

J. Synthesis of CTC Resin-Loaded Dipeptides

Example 112

Loading of Fmoc-MeAla-OH onto CTC Resin

4.47 g of Cl-CTC resin was weighed into a reaction column,dichloromethane (36 mL) was added, and the column was shaken at 30° C.for 60 minutes. After discharging the dichloromethane, a solution ofFmoc-MeAla-OH (1.24 g) and diisopropylethylamine (1.4 mL) indichloromethane (36 mL) was added at room temperature. After stirring at30° C. for three hours, the reaction was analyzed by LCMS to find thatthe reaction conversion rate was 96.0%. After discharging the reactionsolution, a solution of methanol (3.6 mL) and diisopropylethylamine (1.8mL) in DMF (30 mL) was added at room temperature. After shaking at 30°C. for 1.5 hours, the reaction solution was discharged. The resin waswashed with 36 mL of DMF four times and then dried under reducedpressure to give 4.80 g of 55. 56 was also synthesized by the sametechnique. The products were analyzed in the next step.

TABLE 155 Analysis (HPLC method 1)

Reaction Example No. Product CI-CTC resin Amino acid conversion rateProduct amount 112 55 4.47 g 1.24 g 96% 4.80 g 113 10 10.0 g 3.41 g 99%12.4 g

Example 114

Synthesis of Fmoc-MeAla-MeAla-OCTC (22a), Fmoc-MeVal-MeAla-OCTC (23a),and Fmoc-Pro-MeAla-OCTC (24a)

Dipeptides 22a to 24a were simultaneously synthesized using asolid-phase peptide synthesizer Prelude X. 676 mg, 687 mg, and 705 mg ofresin Fmoc-MeAla-OCTC (55) were weighed into three reaction vessels,respectively. DMF (8 mL) was added thereto, and the resin was swollen byallowing to stand at room temperature for one hour. After the DMF wasdischarged, a 20% solution of piperidine in DMF (8 mL) was added and thevessels were shaken at room temperature for 15 minutes. After thesolution was discharged, a 20% solution of piperidine in DMF (8 mL) wasadded again and the vessels were shaken at room temperature for 15minutes. The cocktails 1 to 3 described below were added to RV 1 to RV3, respectively, and a 12.5% solution of DIC in DMF (4 eq.) was thenadded. The vessels were shaken at room temperature for three hours withnitrogen bubbling. After discharging the solution, the resin was washedwith DMF (8 v/w) for 2 min×5 and with MTBE (8 v/w) for 2 min×4, whicheach wash was carried out by shaking at room temperature. The resultingresin was dried under reduced pressure to provide 30a, 31a, and 32a,respectively. The obtained compounds were identified by the resinremoval reactions in Examples 65, 67, and 69.

About 20 mg each of the obtained three resins were weighed into threereaction vessels, a 20% solution of piperidine in DMF (100 mL) was addedto each vessel, and the reaction solution was stirred at roomtemperature for two hours. Dibenzofulvene was quantitatively determinedfrom the absorbance of the solution to calculate the loading rate ofeach dipeptide (Table 156).

Example 115

Synthesis of Fmoc-MeAla-MeAsp(OtBu)-OCTC (25a) andFmoc-MeLeu-MeAsp(OtBu)-OCTC (26a)

Dipeptides 25a to 26a were simultaneously synthesized using asolid-phase peptide synthesizer Prelude X. 340 mg of resinFmoc-MeAsp(OtBu)-OCTC (10) was weighed into each of two reactionvessels. DMF (8 mL) was added thereto, and the resin was swollen byallowing to stand at room temperature for 30 minutes. After the DMF wasdischarged, a 20% solution of piperidine in DMF (8 v/w) was added andthe vessels were shaken at room temperature for 5 minutes. After thesolution was discharged, a 20% solution of piperidine in DMF (8 v/w) wasadded again and the vessels were shaken at room temperature for 20minutes. After discharging the solution, the cocktails 1 and 2 describedbelow were added to the corresponding reaction vessels, respectively, a12.5% solution of DIC in DMF (4 eq.) was then added, and the vesselswere shaken at room temperature for three hours with nitrogen bubbling.After discharging the solution, the resin was washed with DMF (8 v/w)for 2 min×4 and with MTBE (8 v/w) for 2 min×4, which each wash wascarried out by shaking at room temperature. The resulting resin wasdried under reduced pressure to provide 994 mg of 25a and 1.00 g of 26a,respectively. The obtained compounds were identified by the resinremoval reactions in Examples 71 and 73.

About 20 mg each of the obtained two resins were weighed into tworeaction vessels, a 20% solution of piperidine in DMF (100 mL) was addedto each vessel, and the reaction solution was stirred at roomtemperature for two hours. Dibenzofulvene was quantitatively determinedfrom the absorbance of the solution to calculate the loading rate ofeach dipeptide (Table 156).

TABLE 156 Analysis (HPLC method 1) Example Reaction No. Product Rawmaterial conversion rate Loading rate 114 22a 676 mg 100% 0.41 mmol/g23a 687 mg 100% 0.31 mmol/g 24a 705 mg 100% 0.42 mmol/g 115 25a 340 mg100% 0.27 mmol/g 26a 340 mg 100% 0.27 mmol/g

Example 116

Synthesis of Fmoc-MeAsp(OAllyl)-mor

Fmoc-MeAsp(OAllyl)-OH (56, 87.9 g) was added to a reaction vessel anddissolved in DMF (430 ml). HOBt (31.9 g) and EDCI hydrochloride (49.4 g)were then added at ambient temperature, and the reaction solution wascooled to 0° C. Morpholine (20.4 ml) was gradually added to the reactionsolution, which was then stirred at 0° C. for 45 minutes. Water (180 ml)was added at 0° C. to the reaction solution, which was then stirred atambient temperature for one hour. Water (180 ml) was further added andthe reaction solution was stirred at ambient temperature for 1.75 hours.The generated solid was collected by filtration using a Kiriyama funnel,and the resulting solid was washed twice with water (450 ml). The solidafter washing with water was dried under reduced pressure to afford 86.8g (85% yield) of the target product as a colorless solid.

Example 117

Synthesis of Fmoc-MeAsp(OAllyl)—NMe2

Under a nitrogen stream, EDCI hydrochloride (27.4 g) and DMF (217 ml)were added to a reaction vessel, and HOBt (17.7 g) and a solution ofFmoc-MeAsp(OAllyl)-OH (56, 48.8 g) in dichloromethane-DMF (90 ml-90 ml)were then added at 0° C. After stirring the reaction solution at 0° C.for 30 minutes, a dimethylamine-THF solution (2 N, 65.6 ml) was addeddropwise over two minutes to the reaction solution, which was thenstirred at 0° C. for 30 minutes. The reaction solution was diluted withethyl acetate (488 ml), and the organic layer was washed with 1 Nhydrochloric acid (twice with 391 ml), water (488 ml), a 5% aqueoussodium bicarbonate solution (twice with 488 ml), and a 18% aqueoussodium chloride solution (488 ml) and then dried over sodium sulfate.The drying agent was removed by filtration and then the filtrate wasconcentrated under reduced pressure to give 51.2 g (98% yield) of thetarget product as a colorless oil.

Example 118

Synthesis of Fmoc-MeAsp(OH)-mor

Under a nitrogen stream, Fmoc-MeAsp(OAllyl)-mor (57, 22.8 g) anddichloromethane (50 ml) were added to a reaction vessel, andtetrakistriphenylphosphine palladium (0.55 g) was then added at ambienttemperature. Phenylsilane (3.61 g) was added dropwise and the reactionsolution was then stirred at ambient temperature for 30 minutes. Thereaction solution was diluted with MTBE (228 ml) and then extracted witha 5% aqueous sodium bicarbonate solution (228 ml). The aqueous layer wasacidified to about pH 3 with a 85% aqueous phosphoric acid solution (12ml) and extracted with MTBE (228 ml). The organic layer was washed witha 18% aqueous sodium chloride solution (twice with 228 ml) and thendried over sodium sulfate. The drying agent was removed by filtrationand then the filtrate was concentrated under reduced pressure to give20.3 g (97% yield) of the target product as colorless amorphouscrystals.

Example 119

Synthesis of Fmoc-MeAsp(OH)—NMe2

Under a nitrogen stream, Fmoc-MeAsp(OAllyl)-NMe2 (58, 32.0 g) andtetrakistriphenylphosphine palladium (0.847 g) were added to a reactionvessel, and dichloromethane (73.3 ml) was then added. Phenylsilane (5.55g) was added dropwise to the reaction solution, which was then stirredat ambient temperature for 30 minutes. The reaction solution was dilutedwith MTBE (320 ml) and then extracted with a 5% aqueous sodiumbicarbonate solution (308 ml). The aqueous layer was acidified to aboutpH 2 with a 85% aqueous phosphoric acid solution (30.1 ml) and extractedwith MTBE (320 ml). The organic layer was washed with a 18% aqueoussodium chloride solution (twice with 320 ml) and then dried over sodiumsulfate. The drying agent was removed by filtration and then thefiltrate was concentrated under reduced pressure to give 25.1 g (86%yield) of the target product as pale brown amorphous crystals.

TABLE 157 Analysis Compound data Example Retention time No. CompoundHPLC Method MW m/z (min) 116 57 HPLC method D 478.21 479 ([M + H]+)2.570 117 58 HPLC method E 436.20 437 ([M + H]+) 1.262 118 59 HPLCmethod F 438.18 439 ([M + H]+) 0.670 119 60 HPLC method G 396.17 397([M + H]+) 0.680

Example 120

Synthesis of Fmoc-MeAsp(OtBu)-mor (61)

Under a nitrogen stream, Fmoc-MeAsp(OtBu)-OH (39, 3.0 g) and Oxyma (1.1g) were added to a reaction vessel, and dimethylformamide (15 mL) wasthen added. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(1.5 g) and morpholine (0.74 mL) were added to the reaction solutionwhile maintaining the temperature of the reaction solution at 10° C. orlower, and it was stirred for three hours. AcOEt (15 mL) and 0.5 Nhydrochloric acid (15 mL) were added to the reaction solution. Theorganic layer was washed with water (15 mL) and further washed with a 5%aqueous sodium carbonate solution (15 mL). The organic layer was washedwith a 5% aqueous sodium chloride solution (15 mL) and then concentratedunder reduced pressure to afford 3.50 g (100%) of the target product asyellow amorphous crystals. The obtained compound was used for the nextreaction without purification.

Example 121

Synthesis of Fmoc-MeAsp(OtBu)—NMe2 (62)

Under a nitrogen stream, Fmoc-MeAsp(OtBu)-OH (39, 3.0 g) and Oxyma (1.1g) were added to a reaction vessel, and dimethylformamide (15 mL) wasthen added. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(1.5 g), dimethylamine hydrochloride (0.69 g), andN,N-diisopropylethylamine (1.5 ml) were added to the reaction solutionwhile maintaining the temperature of the reaction solution at 10° C. orlower, and it was stirred for three hours. To the reaction solution wasadded ethyl acetate (15 mL), followed by 0.5 N hydrochloric acid (15mL). The organic layer was washed with water (15 mL) and further washedwith a 5% aqueous sodium carbonate solution (15 mL). The organic layerwas washed with a 5% aqueous sodium chloride solution (15 mL) and thenconcentrated under reduced pressure to afford 3.17 g (95%) of the targetproduct as a colorless oily substance. The obtained compound was usedfor the next reaction without purification.

Example 122

Synthesis of Fmoc-MeAsp(OH)-mor (59)

Under a nitrogen stream, Fmoc-MeAsp(OtBu)-mor (61, 1.8 g) and2-methyltetrahydrofuran (9.2 mL) were added to a reaction vessel, andHMDS (0.86 mL) was then added. The reaction vessel was cooled to 0° C.,TMSOTf (0.81 mL) was added, and the reaction solution was stirred at 25°C. for one hour. The reaction vessel was cooled to 0° C., a 5% aqueouspotassium dihydrogenphosphate solution (9.2 mL) was added to thereaction solution, and the organic layer was separated. A 5% aqueoussodium carbonate solution (9.2 mL) was added, and the aqueous layer wasseparated. MTBE (9.2 mL) and 2 N hydrochloric acid (5.0 mL) were addedto the aqueous layer, and the organic layer was separated. The organiclayer was washed with a 5% aqueous potassium dihydrogenphosphatesolution (9.2 mL) and then with a 10% aqueous sodium chloride solution(9.2 mL). The organic layer was concentrated under reduced pressure toafford 1.31 g (97%) of the target product as yellow amorphous crystals.

Example 123

Synthesis of Fmoc-MeAsp(OH)—NMe2 (60)

Under a nitrogen stream, Fmoc-MeAsp(OtBu)—NMe2 (62, 1.6 g) and2-methyltetrahydrofuran (7.9 mL) were added to a reaction vessel, andHMDS (0.80 mL) was then added. The reaction vessel was cooled to 0° C.,TMSOTf (0.75 mL) was added, and the reaction solution was stirred at 25°C. for one hour. A 5% aqueous potassium dihydrogenphosphate solution(7.9 mL) was added to the reaction solution, and the organic layer wasseparated. A 5% aqueous sodium carbonate solution (7.9 mL) was added,and the aqueous layer was separated. MTBE (7.9 mL) and 2 N hydrochloricacid (5.0 mL) were added to the aqueous layer, and the organic layer wasseparated. The organic layer was washed with a 5% aqueous potassiumdihydrogenphosphate solution (7.9 mL) and then with a 10% aqueous sodiumchloride solution (7.9 mL). The organic layer was concentrated underreduced pressure to afford 1.31 g (95%) of the target product as acolorless oily substance.

TABLE 158 Analysis Compound data Example Retention time No. CompoundHPLC Method MW m/z (min) 120 61 HPLC method H 494.24 495.6 ([M + H]+)2.75 121 62 HPLC method H 452.23 453.6 ([M + H]+) 2.77 122 59 HPLCmethod H 438.18 439.5 ([M + H]+) 1.84 123 60 HPLC method H 396.17 397.5([M + H]+) 1.89

INDUSTRIAL APPLICABILITY

The present invention provides methods of producing peptide compounds bynovel deprotection and/or resin removal methods using silylating agents.The present invention can provide industrially applicable and efficienttechniques of peptide synthesis.

1. A method of producing a peptide compound in which a protecting groupremovable by a silylating agent is removed, the method comprising thestep of contacting a starting peptide compound comprising natural aminoacid residues and/or amino acid analog residues with the silylatingagent in a solvent and thereby removing the protecting group from thestarting peptide compound, wherein the silylating agent is prepared bymixing a silyl compound or acid with an electrophilic species scavenger,wherein the starting peptide compound comprises at least one protectinggroup removable by the silylating agent, and wherein the startingpeptide compound comprises at least one N-substituted amino acidresidue.
 2. A method of producing a peptide compound in which a resinfor solid-phase synthesis that is removable by a silylating agent isremoved, the method comprising the step of contacting a starting peptidecompound comprising natural amino acid residues and/or amino acid analogresidues with the silylating agent in a solvent and thereby removing theresin for solid-phase synthesis from the starting peptide compound,wherein the silylating agent is prepared by mixing a silyl compound oracid with an electrophilic species scavenger, wherein the startingpeptide compound is linked to the removable resin for solid-phasesynthesis, and wherein the starting peptide compound comprises at leastone N-substituted amino acid residue.
 3. The method of claim 1 or 2,wherein the starting peptide compound comprises at least one structurein which at least two amino acid residues are linked to each other,wherein the structure is represented by general formula (I) below:

wherein R₁ is hydrogen, PG₁, a natural amino acid residue, or an aminoacid analog residue; R₂ is selected from the group consisting ofhydrogen and C₁-C₆ alkyl, or R₂ and R₄ or R₂ and R_(4′), together withthe nitrogen atom and carbon atom to which they are attached, form a 3-to 7-membered heterocyclic ring optionally substituted with hydroxy orC₁-C₄ alkoxy, wherein R_(4′) is hydrogen when R₂ and R₄ together formthe heterocyclic ring, and R₄ is hydrogen when R₂ and R_(4′) togetherform the heterocyclic ring; except when R₂ and R₄, or R₂ and R_(4T)together form the heterocyclic ring, (a) R_(4′) is hydrogen, and R₄ isselected from the group consisting of hydrogen, optionally substitutedC₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionallysubstituted phenyl, optionally substituted phenylmethyl, optionallysubstituted phenylethyl, 2-(methylthio)ethyl, —CH₂SPG₂,N-PG₃-indol-3-ylmethyl, 4-(PG₂O)benzyl, PG₂-O-methyl, 1-(PG₂O)ethyl,2-(PG₂O)ethyl, PG₂-OCO(CH₂)—, PG₂-OCO(CH₂)₂—, PG₃N-n-butyl,—CON(R_(14A))(R₁₄B), —CH2-CON(R_(14A))(R_(14B)), and—(CH2)2CON(R_(14A))(R_(14B)), (b) R4 and R4′ are independentlyoptionally substituted C1-C6 alkyl, or (c) R4 and R4′, together with thecarbon atom to which they are attached, form a 3-to 7-membered alicyclicring; R₅ is a single bond or —C(R_(5A))(R_(5B))—; R_(5A) and R_(5B) areindependently selected from the group consisting of hydrogen, C₁-C₆alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted aryl-C₁-C₄ alkyl, and optionally substitutedheteroaryl-C₁-C₄ alkyl; R₆ is selected from the group consisting ofhydrogen and C₁-C₆ alkyl, or R₆ and R₇ or R₆ and R_(7′) together withthe nitrogen atom and carbon atom to which they are attached, form a 3-to 7-membered heterocyclic ring optionally substituted with hydroxy orC₁-C₄ alkoxy, wherein R_(7′) is hydrogen when R₆ and R₇ together formthe heterocyclic ring, and R₇ is hydrogen when R₆ and R_(7′) togetherform the heterocyclic ring; except when R₆ and R₇ or R₆ and R_(7′)together form the heterocyclic ring, (a) R_(7′) is hydrogen, and R₇ isselected from the group consisting of hydrogen, optionally substitutedC₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkyl-C₁-C₄ alkyl, optionallysubstituted phenyl, optionally substituted phenylmethyl, optionallysubstituted phenylethyl, 2-(methylthio)ethyl, —CH₂SPG₄,N-PG₅-indol-3-ylmethyl, 4-(PG₄O)benzyl, PG₄-O-methyl, 1-(PG₄O)ethyl,2-(PG₄O)ethyl, PG₄-OCO(CH₂)—, PG₄-OCO(CH₂)₂—, PG₅N-n-butyl,—CON(R_(15A))(R_(15B)), —CH₂—CON(R_(15A))(R_(15B)), and—(CH₂)₂CON(R_(15A))(R_(15B)), or (b) R₇ and R_(7′) are independentlyoptionally substituted C₁-C₆ alkyl, or (c) R₇ and R_(7′), together withthe carbon atom to which they are attached, form a 3- to 7-memberedalicyclic ring; R₈ is a single bond or —C(R_(8A))(R_(8B))—; R_(8A) andR_(8B) are independently selected from the group consisting of hydrogen,C₁-C₆ alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted aryl-C₁-C₄ alkyl, and optionallysubstituted heteroaryl-C₁-C₄ alkyl; R₉ is hydroxy, —O-PG₆, a naturalamino acid residue, an amino acid analog residue, —O-RES, or —NH-RES;RES is a resin for solid-phase synthesis; R_(14A) and R_(14B) areindependently hydrogen or C₁-C₄ alkyl, or R_(14A) and R_(14B), togetherwith the nitrogen atom to which they are attached, form a 4- to8-membered ring optionally comprising one or more additionalheteroatoms; R_(15A) and R_(15B) are independently hydrogen or C₁-C₄alkyl, or R_(15A) and R_(15B), together with the nitrogen atom to whichthey are attached, form a 4- to 8-membered ring optionally comprisingone or more additional heteroatoms; PG₁ is selected from the groupconsisting of Fmoc, Boc, Alloc, Cbz, Teoc, and trifluoroacetyl; PG₂ andPG₄ are independently selected from the group consisting of hydrogen,t-Bu, trityl, methoxytrityl, cumyl, benzyl, THP, 1-ethoxyethyl, methyl,ethyl, allyl, optionally substituted aryl, optionally substitutedaryl-C₁-C₄ alkyl, optionally substituted heteroaryl-C₁-C₄ alkyl, and2-(trimethylsilyl)ethyl; PG₃ and PG₅ are independently selected from thegroup consisting of hydrogen, Fmoc, Boc, Alloc, Cbz, Teoc,methoxycarbonyl, t-Bu, trityl, cumyl, and benzyl; and PG₆ is selectedfrom the group consisting of t-Bu, trityl, cumyl, benzyl, methyl, ethyl,allyl, and 2-(trimethylsilyl)ethyl.
 4. The method of any one of claims 1to 3, wherein the starting peptide compound comprises at the C-terminusa structure in which at least two amino acid residues are linked to eachother, wherein the structure is represented by general formula (II)below:

wherein R_(1′) is a group represented by the formula (III):

* represents the point of attachment; R₁ is hydrogen, PG₁, a naturalamino acid residue, or an amino acid analog residue; R₂ is selected fromthe group consisting of hydrogen and C₁-C₆ alkyl, or R₂ and R₁₀, or R₂and R_(10′), together with the nitrogen atom and carbon atom to whichthey are attached, form a 3- to 7-membered heterocyclic ring optionallysubstituted with hydroxy or C₁-C₄ alkoxy, wherein R_(10′) is hydrogenwhen R₂ and R₁₀ together form the heterocyclic ring, and R₁₀ is hydrogenwhen R₂ and R_(10′) together form the heterocyclic ring; except when R₂and R₁₀ or R₂ and R_(10′) together form the heterocyclic ring, (a)R_(10′) is hydrogen, and R₁₀ is selected from the group consisting ofhydrogen, optionally substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkyl-C₁-C₄ alkyl, optionally substituted phenyl, optionallysubstituted phenylmethyl, optionally substituted phenylethyl,2-(methylthio)ethyl, —CH₂SPG₈, N-PG₉-indol-3-ylmethyl, 4-(PG₈O)benzyl,PG₈-O-methyl, 1-(PG₈O)ethyl, 2-(PG₈O)ethyl, PG₈-OCO(CH₂)—,PG₈-OCO(CH₂)₂—, PG₉N-n-butyl, —CON(R_(16A))(R_(16B)),—CH₂—CON(R_(16A))(R_(16B)), and —(CH₂)₂CON(R_(16A))(R_(16B)), or (b) R₁₀and R_(10′) are independently optionally substituted C₁-C₆ alkyl, C₃-C₆cycloalkyl, or C₃-C₆ cycloalkyl-C₁-C₄ alkyl, or (c) R₁₀ and R_(10′),together with the carbon atom to which they are attached, form a 3- to7-membered alicyclic ring; R₁₁ is a single bond or—C(R_(11A))(R_(11B))—; R_(11A) and R_(11B) are independently selectedfrom the group consisting of hydrogen, C₁-C₆ alkyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted aryl-C₁-C₄ alkyl, and optionally substitutedheteroaryl-C₁-C₄ alkyl; R₁₂ and R_(12′) are independently selected fromthe group consisting of hydrogen, PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀,—(CH₂)_(n)COO-RES, and —(CH₂)_(n)CONH-RES; RES is a resin forsolid-phase synthesis; n is 0, 1, or 2; R₆ is selected from the groupconsisting of hydrogen and C₁-C₆ alkyl; R₁₃ is C₁-C₄ alkyl or—(CH₂)_(m)CON(R_(17A))(R_(17B)); m is 0, 1, or 2; R_(16A) and R_(16B)are independently hydrogen or C₁-C₄ alkyl, or R_(16A) and R_(16B),together with the nitrogen atom to which they are attached, form a 4- to8-membered ring optionally comprising one or more additionalheteroatoms; R_(17A) and R_(17B) are independently hydrogen or C₁-C₄alkyl, or R_(17A) and R_(17B), together with the nitrogen atom to whichthey are attached, form a 4- to 8-membered ring optionally comprisingone or more additional heteroatoms; PG₁ is independently selected fromthe group consisting of Fmoc, Boc, Alloc, Cbz, Teoc, andtrifluoroacetyl; PG₈ is selected from the group consisting of hydrogen,t-Bu, trityl, methoxytrityl, cumyl, benzyl, THP, 1-ethoxyethyl, methyl,ethyl, allyl, optionally substituted aryl, optionally substitutedaryl-C₁-C₄ alkyl, optionally substituted heteroaryl-C₁-C₄ alkyl, and2-(trimethylsilyl)ethyl; PG₉ is selected from the group consisting ofhydrogen, Fmoc, Boc, Alloc, Cbz, Teoc, methoxycarbonyl, t-Bu, trityl,cumyl, and benzyl; and PG₁₀ is selected from the group consisting oft-Bu, trityl, cumyl, benzyl, methyl, ethyl, allyl, optionallysubstituted aryl, optionally substituted aryl-C₁-C₄ alkyl, optionallysubstituted heteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl.
 5. Amethod of producing an amide compound in which a protecting groupremovable by a silylating agent is removed, the method comprising thestep of contacting a starting amide compound with the silylating agentin a solvent and thereby removing the protecting group from the startingamide compound, wherein the silylating agent is prepared by mixing asilyl compound or acid with an electrophilic species scavenger, whereinthe starting amide compound is represented by general formula (II)below:

wherein R_(1′) is a hydrogen atom or PG₇; R₁₂ and R_(12′) areindependently selected from the group consisting of hydrogen,PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀, —(CH₂)_(n)COO-RES, and—(CH₂)_(n)CONH-RES; RES is a resin for solid-phase synthesis; n is 0, 1,or 2; R₆ is selected from the group consisting of hydrogen and C₁-C₆alkyl; R₁₃ is C₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B)); m is 0, 1,or 2; R_(17A) and R_(17B) are independently hydrogen or C₁-C₄ alkyl, orR_(17A) and R_(17B), together with the nitrogen atom to which they areattached, form a 4- to 8-membered ring optionally comprising one or moreadditional heteroatoms; PG₇ is selected from the group consisting ofFmoc, Boc, Alloc, Cbz, Teoc, and trifluoroacetyl; and PG₁₀ is selectedfrom the group consisting of t-Bu, trityl, cumyl, benzyl, methyl, ethyl,allyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄alkyl, optionally substituted heteroaryl-C₁-C₄ alkyl, and2-(trimethylsilyl)ethyl, and wherein the starting amide compoundcomprises at least one protecting group removable by the silylatingagent.
 6. A method of producing an amide compound in which a resin forsolid-phase synthesis is removed, the method comprising the step ofcontacting a starting amide compound with a silylating agent in asolvent and thereby removing the starting amide compound from the resinfor solid-phase synthesis, wherein the silylating agent is prepared bymixing a silyl compound or acid with an electrophilic species scavenger,and wherein the starting amide compound is represented by generalformula (II) below:

wherein R_(1′) is a hydrogen atom or PG₇; R₁₂ and R_(12′) areindependently selected from the group consisting of hydrogen,PG₁₀-O-methyl, —(CH₂)_(n)COO-PG₁₀, —(CH₂)_(n)COO-RES, and—(CH₂)_(n)CONH-RES; RES is a resin for solid-phase synthesis, wherein atleast one of R₁₂ and R_(12′) is —(CH₂)_(n)COO-RES or —(CH₂)_(n)CONH-RES;RES is a resin for solid-phase synthesis; n is 0, 1, or 2; R₆ isselected from the group consisting of hydrogen and C₁-C₆ alkyl; R₁₃ isC₁-C₄ alkyl or —(CH₂)_(m)CON(R_(17A))(R_(17B)); m is 0, 1, or 2; R_(17A)and R_(17B) are independently hydrogen or C₁-C₄ alkyl, or R_(17A) andR_(17B), together with the nitrogen atom to which they are attached,form a 4- to 8-membered ring optionally comprising one or moreadditional heteroatoms; PG₇ is selected from the group consisting ofFmoc, Boc, Alloc, Cbz, Teoc, and trifluoroacetyl; and PG₁₀ is selectedfrom the group consisting of t-Bu, trityl, cumyl, benzyl, methyl, ethyl,allyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄alkyl, optionally substituted heteroaryl-C₁-C₄ alkyl, and2-(trimethylsilyl)ethyl.
 7. The method of any one of claims 1 and 3 to5, wherein the removable protecting group is selected from the groupconsisting of t-Bu, triphenylmethyl, 2-(trimethylsilyl)-ethyl, Boc,Teoc, Cbz, methoxycarbonyl, tetrahydropyranyl, 1-ethoxyethyl,methoxytrityl, and cumyl.
 8. The method of any one of claims 1 to 7,wherein the silyl compound is represented by formula 1 below:

wherein R_(AX), R_(AY), and R_(AZ) are independently C₁-C₄ alkyl orphenyl, and X is selected from the group consisting of —OTf, —OClO₃, Cl,Br, and I.
 9. The method of claim 8, wherein the silyl compound isselected from the group consisting of TMSOTf, TESOTf, TBSOTf, TIPSOTf,TBDPSOTf, TTMSOTf, TMSCl, TMSBr, TMSOClO₃, and TMSI.
 10. The method ofany one of claims 1 to 7, wherein the acid is represented by HX, whereinX is selected from the group consisting of —OTf, —OClO₃, Cl, Br, and I.11. The method of any one of claims 1 to 10, wherein the electrophilicspecies scavenger is selected from the group consisting of formulas (2)to (10) below:

wherein in formula 2, R_(B) is a substituted silyl group and R_(C) is asubstituted silyl group, or R_(B) and R_(C), together with the nitrogenatom and carbon atom to which they are attached, form a 5- to 7-memberedring; and R_(D) is C₁-C₄ alkyl optionally substituted with one or morefluorine atoms or is optionally substituted methylene, wherein whenR_(D) is optionally substituted methylene, formula 2 is dimerized toform a compound represented by the formula below:

wherein in formula 3, R_(G) is a silyl group substituted with one ormore C₁-C₄ alkyl; R_(H) is hydrogen or C₁-C₄ alkyl; and R_(I) ishydrogen, or C₁-C₄ alkyl optionally substituted with one or morefluorine atoms; wherein in formula 4, (a-1) R_(J) is a substituted silylgroup, R_(K) is C₁-C₄ alkoxy, and R_(M) and R_(L) are independentlyhydrogen or C₁-C₄ alkyl; (a-2) R_(J) is a substituted silyl group, R_(M)is hydrogen or C₁-C₄ alkyl, and R_(K) and R_(L), together with thecarbon atoms to which they are attached, form a 5- to 8-membered ringcomprising an oxygen atom; (b-1) R_(J) is a substituted silyl group,R_(K) is C₁-C₄ alkyl, and R_(M) and R_(L) are independently hydrogen orC₁-C₄ alkyl; (b-2) R_(J) is a substituted silyl group, R_(M) is hydrogenor C₁-C₄ alkyl, and R_(K) and R_(L) are taken together with the carbonatoms to which they are attached, form a 5- to 8-membered ring; or (c-1)RJ and RM, together with the carbon atoms to which they are attached,form a 5- to 7-membered ring comprising an oxygen atom, RK is hydrogenor C1-C4 alkyl, and RL is C1-C4 alkyl; (c-2) RJ and RM, together withthe carbon atoms to which they are attached, form a 5- to 7-memberedring comprising an oxygen atom, and RK and RL, together with the carbonatom to which they are attached, form a 5- to 8-membered ring; (d-1) RJis C1-C4 alkyl and RM, RK, and RL are independently hydrogen or C1-C4alkyl; (d-2) RJ is C1-C4 alkyl, RM is hydrogen or C1-C4 alkyl, and RKand RL, together with the carbon atoms to which they are attached, forma 5- to 8-membered ring; (e-1) RJ is C1-C3 alkylcarbonyl and RM, RK, andRL are independently hydrogen or C1-C4 alkyl; (e-2) RJ is C1-C3alkylcarbonyl, RM is hydrogen or C1-C4 alkyl, and RK and RL, togetherwith the carbon atoms to which they are attached, form a 5- to8-membered ring; (f-1) RJ is a substituted silyl group or C1-C4 alkyl,RK is optionally substituted di-C1-C4 alkylamino, and RM and RL areindependently hydrogen or C1-C4 alkyl; or (f-2) RJ is a substitutedsilyl group or C1-C4 alkyl, RM is hydrogen or C1-C4 alkyl, and RK andRL, together with the carbon atoms to which they are attached, form a 5-to 8-membered ring comprising a nitrogen atom, wherein the 5- to8-membered ring is optionally substituted with C₁-C₄ alkyl; wherein informula 5, R_(N), R_(N′), and R_(O) are independently hydrogen or C₁-C₄alkyl; wherein in formula 6, R_(P) is a substituted silyl group; andR_(Q) is a substituted silyl group or C₁-C₄ alkyl and R_(R) is hydrogen,a substituted silyl group, or C1-C4 alkyl, or R_(Q) and R_(R), togetherwith the nitrogen atom to which they are attached, form a 5- to8-membered heterocyclic ring optionally comprising one or moreadditional heteroatoms; wherein in formula 7, X is a single bond or acarbon atom, wherein when X is a single bond, R_(S) is absent, R_(UA)and R_(R), together with the carbon atom and nitrogen atom to which theyare attached, form an optionally substituted 6-membered aromaticheterocyclic ring, and R_(UB) and R_(T), together with the carbon atomand nitrogen atom to which they are attached, form an optionallysubstituted 6-membered aromatic heterocyclic ring, and when X is acarbon atom, R_(UA) and R_(UB) are independently C₁-C₄ alkyl and R_(R),R_(S), and R_(T), together with the carbon atoms to which they areattached, form the structure below:

wherein in formula 8, R_(V) is C₁-C₄ alkyl or C₃-C₆ cycloalkyl; whereinin formula 9, R_(W) and R_(X) are independently C₁-C₄ alkyl or asubstituted silyl group; and wherein in formula 10, R_(Y) and R_(Z) areindependently C₁-C₄ alkyl or a substituted silyl group.
 12. The methodof claim 11, wherein the electrophilic species scavenger is selectedfrom the group consisting of N,O-bis(trimethylsilyl)acetamide,N,O-bis(trimethylsilyl)trifluoroacetamide,N-methyl-N-trimethylsilylacetamide,N-methyl-N-trimethylsilyltrifluoroacetamide, dimethylketene methyltrimethylsilyl acetal, isopropenyloxytrimethylsilane,2,2,4,4-tetramethylpentanone imine, 1,1,1,3,3,3-hexamethyldisilazane(HMDS), N-trimethylsilylmorpholine, N-trimethylsilyldiethylamine, andN-tert-butyltrimethylsilylamine.
 13. The method of any one of claims 1to 9 and 11 to 12, wherein per one equivalent of the protecting group tobe removed or one equivalent of the resin to be removed, 1 to 5equivalents of the silyl compound and 1 to 10 equivalents of theelectrophilic species scavenger are mixed.
 14. The method of any one ofclaims 1 to 12, wherein per one equivalent of the protecting group to beremoved or one equivalent of the resin to be removed, 0.1 to 0.5equivalent of the silyl compound or acid is mixed, wherein theelectrophilic species scavenger is selected from the group consisting ofN,O-bis(trimethylsilyl)acetamide,N,O-bis(trimethylsilyl)trifluoroacetamide,N-methyl-N-trimethylsilylacetamide,N-methyl-N-trimethylsilyltrifluoroacetamide, dimethylketene methyltrimethylsilyl acetal, and isopropenyloxytrimethylsilane, wherein thesilyl compound is selected from the group consisting of TMSOTf, TESOTf,TBSOTf, TIPSOTf, TBDPSOTf, TTMSOTf, TMSCl, TMSBr, and TMSOClO₃, andwherein the acid is represented by HX, wherein X is selected from thegroup consisting of —OTf, —OClO₃, Cl, Br, and I.
 15. The method of anyone of claims 1 to 14, wherein the starting peptide compound comprises 1to 30 amino acid residues and is linear or cyclic.
 16. The method of anyone of claims 1 to 4 and 6 to 15, wherein the resin for solid-phasesynthesis is CTC resin, Wang resin, or SASRIN resin.
 17. The method ofany one of claims 1 to 16, wherein the method comprises mixing thestarting peptide compound with the solvent, then with the electrophilicspecies scavenger, and subsequently with the silyl compound or acid. 18.An amide compound represented by general formula (A) below or a saltthereof:

wherein R_(1′) is selected from the group consisting of hydrogen, Fmoc,Boc, Alloc, Cbz, Teoc, and trifluoroacetyl; R_(17A) and R_(17B) are bothmethyl, or R_(17A) and R_(17B), together with the nitrogen atom to whichthey are attached, form piperidine or morpholine; and R₁₈ is hydrogen orPG₁₀, wherein PG₁₀ is selected from the group consisting of t-Bu,trityl, cumyl, benzyl, methyl, ethyl, allyl, optionally substitutedaryl, optionally substituted aryl-C₁-C₄ alkyl, optionally substitutedheteroaryl-C₁-C₄ alkyl, and 2-(trimethylsilyl)ethyl.
 19. The amidecompound or salt thereof of claim 18, wherein the amide compound isselected from the group consisting of: (3-1)3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoicacid, (3-2) allyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-3) tert-butyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-4) benzyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-5)3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoicacid, (3-6) allyl3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-7) tert-butyl3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-8) benzyl3-((tert-butoxycarbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-9)3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoicacid, (3-10) allyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-11) tert-butyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-12) benzyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-13)3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoicacid, (3-14) allyl3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-15) tert-butyl3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-16) benzyl3-(((allyloxy)carbonyl)(methyl)amino)-4-(dimethylamino)-4-oxobutanoate,(3-17)4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoicacid, (3-18) allyl4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,(3-19) tert-butyl4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,(3-20) benzyl4-(dimethylamino)-3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxobutanoate,(2-1)3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoicacid, (2-2) allyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-3) tert-butyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-4) benzyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-5) 3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoicacid, (2-6) allyl3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-7) tert-butyl3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-8) benzyl3-((tert-butoxycarbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-9) 3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoicacid, (2-10) allyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-11) tert-butyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-12) benzyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-13) 3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoicacid, (2-14) allyl3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-15) tert-butyl3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-16) benzyl3-(((allyloxy)carbonyl)(methyl)amino)-4-morpholino-4-oxobutanoate,(2-17)3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoicacid, (2-18) allyl3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,(2-19) tert-butyl3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,(2-20) benzyl3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-morpholino-4-oxobutanoate,(4-1) 3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoic acid, (4-2) allyl3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate, (4-3) tert-butyl3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate, (4-4) benzyl3-(methylamino)-4-oxo-4-(piperidin-1-yl)butanoate, (4-5)3-(methylamino)-4-morpholino-4-oxobutanoic acid, (4-6) allyl3-(methylamino)-4-morpholino-4-oxobutanoate, (4-7) tert-butyl3-(methylamino)-4-morpholino-4-oxobutanoate, (4-8) benzyl3-(methylamino)-4-morpholino-4-oxobutanoate, (4-9)4-(dimethylamino)-3-(methylamino)-4-oxobutanoic acid, (4-10) allyl4-(dimethylamino)-3-(methylamino)-4-oxobutanoate, (4-11) tert-butyl4-(dimethylamino)-3-(methylamino)-4-oxobutanoate, (4-12) benzyl4-(dimethylamino)-3-(methylamino)-4-oxobutanoate, (1-1)3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoicacid, (1-2) allyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-3) tert-butyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-4) benzyl3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-5)3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoicacid, (1-6) allyl3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-7) tert-butyl3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-8) benzyl3-((tert-butoxycarbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-9)3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoicacid, (1-10) allyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-11) tert-butyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-12) benzyl3-(((benzyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-13)3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoicacid, (1-14) allyl3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-15) tert-butyl3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-16) benzyl3-(((allyloxy)carbonyl)(methyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-17)3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoicacid, (1-18) allyl3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,(1-19) tert-butyl3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate,and (1-20) benzyl3-(methyl((2-(trimethylsilyl)ethoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate.